CN114728886A - Carbonate-containing lipid compounds and compositions for intracellular delivery of therapeutic agents - Google Patents

Abstract

The present application relates to lipids of general formula (a), wherein at least one of the two side chains contains a carbonate functional group (M and/or M' is-OC (═ O) O-), and to empty or loaded Lipid Nanoparticles (LNPs) comprising such lipids as well as further lipids, such as phospholipids, structural lipids and PEG lipids. Lipid nanoparticles that also comprise therapeutic and/or prophylactic agents, such as RNA, can be used to deliver therapeutic and/or prophylactic agents to mammalian cells or organs to, for example, regulate polypeptide, protein, or gene expression.

Description

Carbonate-containing lipid compounds and compositions for intracellular delivery of therapeutic agents

Related applications

This application claims priority to and the benefit of US Provisional Application No. 62/902,929, filed September 19, 2019, the entire disclosure of which is incorporated herein by reference.

technical field

The present disclosure provides novel compounds, compositions comprising such compounds, and compositions comprising lipid nanoparticles for the delivery of one or more therapeutic and/or prophylactic agents to and/or in mammalian cells or organs or a method of producing a polypeptide in an organ. In addition to novel lipids, lipid nanoparticle compositions of the present disclosure may also include specified fractions of one or more cationic and/or ionizable amino lipids, phospholipids including polyunsaturated lipids , PEG lipids, structured lipids, and/or therapeutic and/or prophylactic agents.

Background technique

Efficient targeted delivery of biologically active substances such as small molecule drugs, proteins and nucleic acids presents an enduring medical challenge. In particular, delivery of nucleic acids to cells is made difficult by the relative instability and low cellular permeability of such substances. Accordingly, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and/or prophylactic agents, such as nucleic acids, to cells.

Lipid-containing nanoparticle compositions, liposomes and liposome complexes have been shown to be effective as transport vehicles for biologically active substances such as small molecule drugs, proteins and nucleic acids into cells and/or intracellular compartments. Such compositions typically comprise one or more "cationic" and/or amino (ionizable) lipids, phospholipids including polyunsaturated lipids, structured lipids (eg, sterols) and/or Lipids containing polyethylene glycol (PEG lipids). Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated. Although a variety of such lipid-containing nanoparticle compositions have been demonstrated, improvements in safety, efficacy and specificity are still lacking.

SUMMARY OF THE INVENTION

The present disclosure provides novel compounds and compositions and methods including the same.

In some aspects, the present disclosure relates to compounds of formula (A):

或其N-氧化物，或它们的盐或异构体，其中

or their N-oxides, or their salts or isomers, wherein

m is selected from 5, 6, 7, 8 and 9;

R ² and R ³ are each independently selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

M and M' are each independently selected from -OC(O)O- and -C(O)O-;

wherein at least one of M and M' is -OC(O)O-;

R' ^a is C _1-18 alkyl or C _2-18 alkenyl; and

R" is C3 _{- C13} alkyl optionally substituted with OH.

Detailed ways

The present disclosure relates to novel lipids and lipid nanoparticles comprising novel lipids (eg, empty LNPs or loaded LNPs). The present disclosure also provides methods of delivering therapeutic and/or prophylactic agents to mammalian cells, in particular, delivering therapeutic and/or prophylactic agents to mammalian organs in which a polypeptide of interest is produced, Increasing protein levels produced in mammalian cells compared to LNPs comprising other lipids, and treating a disease or disorder in a mammal in need thereof. For example, a method of producing a polypeptide of interest in a cell includes contacting a nanoparticle comprising mRNA with a mammalian cell, whereby the mRNA can be translated to produce the polypeptide of interest. A method of delivering a therapeutic and/or prophylactic agent to a mammalian cell or organ may comprise administering to a subject a nanoparticle composition comprising the therapeutic and/or prophylactic agent, wherein the administering comprises subjecting the cell or organ to the combination contact, thereby delivering therapeutic and/or prophylactic agents to cells or organs. Such delivery methods can be in vitro or in vivo.

The present disclosure provides lipids comprising a central amine moiety and at least one biodegradable group. The lipids described herein can be advantageously used in lipid nanoparticle compositions to deliver therapeutic and/or prophylactic agents to mammalian cells or organs. For example, the lipids described herein have little or no immunogenicity. For example, formulas (A), (A1), (A2), (A3), (B), (I), (I'), (I-X) compared to a reference lipid (eg, MC3, KC2, or DLinDMA) ), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) Or the lipid compounds of (IIb) are less immunogenic. For example, a formulation comprising a lipid disclosed herein and a therapeutic or prophylactic agent has an increased therapeutic index compared to a corresponding formulation comprising a reference lipid (eg, MC3, KC2 or DLinDMA) and the same therapeutic or prophylactic agent.

其中In some aspects, the present disclosure relates to compounds of formula (B):

in

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

R ^aα , R ^aβ and R ^aγ are each independently selected from the group consisting of H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^aα , R ^aβ and R ^aγ is selected from C _1- the group consisting of ₁₂ alkyl and C _2-12 alkenyl; and

R' is C _1-12 alkyl or C _2-12 alkenyl.

In some aspects, the present disclosure relates to compounds of formula (A1 ), (A2), or (A3):

in

m is selected from 5, 6, 7, 8 and 9;

R ² and R ³ are each independently selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

^R'a is:

R ^aα , R ^aβ and R ^aγ are each independently selected from the group consisting of H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^aα , R ^aβ and R ^aγ is selected from C _1-12 the group consisting of alkyl and C _2-12 alkenyl;

R" is C3 _{- C13} alkyl optionally substituted with OH; and

R' is C _1-12 alkyl or C _2-12 alkenyl.

In some aspects, the present disclosure relates to compounds of formula (I):

或其N-氧化物，

or its N-oxide,

or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M is selected from -OC(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -OC(O)N(R ^M )-, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, SO-, -OS-, -S(R ^M ) ₂ O-, -OS( R ^M ) ₂ -, -S(O)O-, -OS(O)-, wherein M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) _2- , -S(R**) _2- or -S(O)-, wherein z is 1, 2, 3 or 4;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of _C1-6 alkyl, _C1-3 alkyl-aryl, _C2-3 alkenyl, and H;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some aspects, the present disclosure relates to compounds of formula (I'):

或其N-氧化物，

or its N-oxide,

or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M )- , -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, - C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, - OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, - (CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R** ) _2- or -S(O)-, wherein z is 1, 2, 3, or 4;

M' is selected from -OC(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -OC(O)N(R ^M )-, -N( R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, SO-, -OS-, -S(R ^M ) ₂ O-, -OS (R ^M ) ₂ -, -S(O)O-, -OS(O)-, wherein M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _{2- 13Alkenyl} , -B(R**)-, -Si(R**) _2- , -S(R**) _2- or -S(O)-, wherein z is 1, 2, 3, or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle;

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some aspects, the present disclosure relates to compounds of formula (I-X):

或其N-氧化物，或它们的盐或异构体，其中

or their N-oxides, or their salts or isomers, wherein

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In certain embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (IA):

or an N-oxide thereof, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4 and 5; m is selected from 5, 6, 7, 8 and ⁹ ; and R and ^R are independently is selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl.

In certain embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (II):

or N-oxides thereof, or their salts or isomers, wherein I is selected from 1, 2, 3, 4 and 5; and R ² and R ³ are independently selected from H, C _1-14 alkyl and C _{2 -14} group consisting of alkenyl groups.

In other embodiments, the subset of compounds of formula (II) includes compounds of formula (IIa):

or their N-oxides, or their salts or isomers, wherein

l is selected from 1, 2, 3, 4 and 5;

M ₁ is M'; and

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl;

In other embodiments, the subset of compounds of formula (II) includes compounds of formula (IIb):

or their N-oxides, or their salts or isomers, wherein

l is selected from 1, 2, 3, 4 and 5;

M ₁ is M'; and

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl.

In other embodiments, the subset of compounds of Formula (I) or Formula (I') includes compounds of Formula (Ia):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (Ib):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (Ic):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (Id):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (Ie):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (If):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In other embodiments, the subset of compounds of formula (I) or formula (I') includes compounds of formula (Ig):

或其N-氧化物，或它们的盐或异构体，其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

In some aspects, the present disclosure relates to compounds of formula (I-Y):

或其N-氧化物，或它们的盐或异构体，其中：

or their N-oxides, or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In other embodiments, the subset of compounds of formula (I-Y) includes compounds of formula (I-Ya):

或其N-氧化物，或它们的盐或异构体，其中每个R”独立地选自H、OH和C_1-6烷基，l选自2、3、4、5和6，并且m选自5、6、7、8和9。

or an N-oxide thereof, or a salt or isomer thereof, wherein each R" is independently selected from H, OH, and C _1-6 alkyl, and l is selected from 2, 3, 4, 5, and 6, and m is selected from 5, 6, 7, 8 and 9.

Formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia) Compounds of any one of , (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) when applicable include one of the following features or more.

其中

表示连接点；In some embodiments, ^R'a is:

in

represents a connection point;

R ^aα , R ^aβ and R ^aγ are each independently selected from the group consisting of H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^aα , R ^aβ and R ^aγ is selected from C _1- the group consisting of ₁₂ alkyl and C _2-12 alkenyl; and

R' is the group consisting of C _1-12 alkyl or C _2-12 alkenyl.

In some embodiments, M and M' are each -OC(O)O-. In some embodiments, M is -C(O)O- and M' is -OC(O)O-. In some embodiments, M is -OC(O)O- and M is -C(O)O-.

In some embodiments, 1 is 1, 3, or 5. In some embodiments, 1 is 1, 3, or 4. In some embodiments, 1 is 5.

In some embodiments, R ⁴ is

In some embodiments, R ⁴ is -(CH ₂ ) ₂ OH.

In some embodiments, R ² and R ³ are independently C _3-14 alkyl or C _3-14 alkenyl.

In some embodiments, ^R'a is selected from _C4 alkyl, _C4 alkenyl, _C5 alkyl, _C5 alkenyl, _C6 alkyl, _C6 alkenyl, _C7 alkyl, _C7 alkenyl , _C9 alkyl, _C9 alkenyl, _C11 alkyl, _C11 alkenyl, _C17 alkyl, _C17 alkenyl, _C18 alkyl and _C18 alkenyl, each of which is straight or branched .

In some embodiments, R' is _C4 alkyl or _C4 alkenyl. In some embodiments, R' is _C5 alkyl or _C5 alkenyl. In some embodiments, R' is _C6 alkyl or _C6 alkenyl. _In some embodiments, R' is C7 alkyl or _C7 alkenyl. _In some embodiments, R' is C8 alkyl or _C8 alkenyl. In some embodiments, R' is _C9 alkyl or _C9 alkenyl. In some embodiments, R' is _C10 alkyl or _C10 alkenyl. In some embodiments, R' is C ₁₁ alkyl or C ₁₁ alkenyl.

In certain embodiments, ^R'a is branched C _1-18 alkyl. For example, ^R'a is

In some embodiments, R" is independently selected from the group consisting of _C3-15 alkyl and _C3-15 alkenyl. In some embodiments, _R " is C3 alkyl, _C4 alkyl, _C5 alkyl, _C6 alkyl, _C7 alkyl or _C8 alkyl. In some embodiments, R" is _C9 alkyl, _C10 alkyl, _C11 alkyl, _{C12 alkyl, C13 alkyl, C14 alkyl ,} or _C15 alkyl.

In some embodiments, R ² and R ³ are independently C _5-14 alkyl or C _5-14 alkenyl.

In some embodiments, R ² is the same as R ³ . In some embodiments, R ² and R ³ are C ₈ alkyl. In certain embodiments, R ² and R ³ are C ₂ alkyl. In other embodiments, R ² and R ³ are C ₃ alkyl. In some embodiments, R ² and R ³ are C ₄ alkyl. In certain embodiments, R ² and R ³ are C ₅ alkyl. In other embodiments, R ² and R ³ are C ₆ alkyl. In some embodiments, R ² and R ³ are C ₇ alkyl.

In other embodiments, R ² is different from R ³ . In certain embodiments, R ² is C ₈ alkyl. In some embodiments, R ³ is C _1-7 (eg, C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , or C ₇ alkyl) or C ₉ alkyl.

In some embodiments, R ³ is C ₁ alkyl. In some embodiments, R ³ is C ₂ alkyl. In some embodiments, R ³ is C ₃ alkyl. In some embodiments, R ³ is C ₄ alkyl. In some embodiments, R ³ is C ₅ alkyl. In some embodiments, R ³ is C ₆ alkyl. In some embodiments, R ³ is C ₇ alkyl. In some embodiments, R ³ is C ₉ alkyl.

In some embodiments, m is 5, 6, 7, 8, or 9. In some embodiments, m is 5, 7 or 9. For example, in some embodiments, m is 5. For example, in some embodiments, m is 7. For example, in some embodiments, m is 9.

In some embodiments, n is 2, 3, or 4. In some embodiments, n is 2. In some embodiments, n is 4. In some embodiments, n is not 3.

In some embodiments, n2 is 2, 3, or 4. In some embodiments, n2 is 2. In some embodiments, n2 is 4. In some embodiments, n2 is not 3.

In some embodiments, formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) ( The compounds of IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) are selected from Table 1.

Table 1: Amino lipids

According to formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia ), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) The central amine moiety of the lipid can be protonated at physiological pH change. Thus, lipids can be positively or partially positively charged at physiological pH. Such lipids may be referred to as cationic or ionizable (amino) lipids. Lipids can also be zwitterionic, ie, neutral molecules that are both positively and negatively charged.

definition

As used herein, the term "alkyl" or "alkyl group" means comprising one or more carbon atoms (eg, one, two, three, four, five, six, seven, eight , nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) optionally substituted straight-chain or branched-chain saturated hydrocarbons. The designation "C _1-14 alkyl" means an optionally substituted straight or branched chain saturated hydrocarbon containing 1 to 14 carbon atoms. Unless otherwise specified, alkyl groups described herein refer to both unsubstituted and substituted alkyl groups.

As used herein, the term "alkenyl" or "alkenyl group" means a group comprising two or more carbon atoms (eg, two, three, four, five, six, seven, eight , nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and optionally substituted straight or branched chain hydrocarbons with at least one double bond. The designation "C _2-14 alkenyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon double bond. Alkenyl groups can contain one, two, three, four or more carbon-carbon double bonds. For example, a _C18 alkenyl group may contain one or more double bonds. A _C18 alkenyl group containing two double bonds may be a linoleyl group. Unless otherwise specified, alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups.

As used herein, the term "alkynyl" or "alkynyl group" means a group containing two or more carbon atoms (eg, two, three, four, five, six, seven, eight , nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond optionally substituted straight or branched chain hydrocarbon. The designation "C _2-14 alkynyl" means an optionally substituted straight or branched chain hydrocarbon containing 2 to 14 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups can contain one, two, three, four or more carbon-carbon triple bonds. For example, a _C18 alkynyl group may contain one or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups.

As used herein, the term "carbocycle" or "carbocyclic group" means an optionally substituted monocyclic or polycyclic ring system comprising one or more rings composed of carbon atoms. Rings can be three yuan, four yuan, five yuan, six yuan, seven yuan, eight yuan, nine yuan, ten yuan, eleven yuan, twelve yuan, thirteen yuan, fourteen yuan, fifteen yuan, sixteen yuan , seventeen, eighteen, nineteen or twenty ring. The designation " _C3-6 carbocycle" means a carbocycle comprising a monocyclic ring having 3-6 carbon atoms. Carbocycles can contain one or more carbon-carbon double or triple bonds, and can be non-aromatic or aromatic rings (eg, cycloalkyl or aryl). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl. As used herein, the term "cycloalkyl" means a non-aromatic carbocyclic ring and may or may not contain any double or triple bonds. Unless otherwise specified, carbocycles described herein refer to both unsubstituted and substituted carbocyclic groups, ie, optionally substituted carbocycles. In some embodiments, the carbocycle is C _3-8 cycloalkyl. In some embodiments, the carbocycle is C _3-6 cycloalkyl. In some embodiments, the carbocycle is a _C6-10 aryl.

"Aryl" includes groups having aromaticity, including "conjugated" or polycyclic systems having at least one aromatic ring and not containing any heteroatoms in the ring structure. Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthyl, and the like. In some embodiments, "aryl" is a _C6-10 carbocyclic ring having aromaticity (eg, "aryl" is a _C6-10 aryl group).

As used herein, the term "heterocycle" or "heterocyclic group" means an optionally substituted monocyclic or polycyclic ring system comprising one or more rings, at least one ring comprising at least one heteroatom. Heteroatoms can be, for example, nitrogen, oxygen or sulfur atoms. Rings can be three-, four-, five-, six-, seven-, eight-, nine-, ten-, eleven-, twelve-, thirteen-, or fourteen-membered rings. Heterocycles may contain one or more double or triple bonds and may be non-aromatic or aromatic rings (eg, heterocycloalkyl or heteroaryl). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazole Alkyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuranyl, thienyl, pyridyl, piperidinyl, quinolinyl and isoquinolinyl. As used herein, the term "heterocycloalkyl" means a non-aromatic heterocycle and may or may not contain any double or triple bonds. Unless otherwise specified, heterocycles described herein refer to both unsubstituted and substituted heterocycle groups, ie, optionally substituted heterocycles. In some embodiments, the heterocycle is a 4- to 12-membered heterocycloalkyl. In some embodiments, the heterocycle is a 5- or 6-membered heteroaryl.

"Heteroaryl" is an aryl group as defined above, except having one to four heteroatoms in the ring structure, and may also be referred to as an "arylheterocycle" or "heteroaromatic". As used herein, the term "heteroaryl" is intended to encompass stable 5, 6 or 7 membered monocyclic or 7, 8, 9, 10, 11 or 12 membered bicyclic aromatic heterocycles consisting of carbon atoms and one or more heteroatoms such as 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms independently selected from the group consisting of nitrogen, oxygen sulfur and boron, or For example 1, 2, 3, 4, 5 or 6 heteroatoms. Nitrogen atoms can be substituted or unsubstituted (ie, N or NR, where R is H or other substituents as defined). Nitrogen and sulfur heteroatoms can be optionally oxidized (ie, N→O and S(O) _p , where p=1 or 2). Note that the total number of S and O atoms in the aromatic heterocycle does not exceed one.

Examples of heteroaryl groups include pyrrole, furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

In addition, the terms "aryl" and "heteroaryl" include polycyclic aryl and heteroaryl groups, such as tricyclic, bicyclic, such as naphthalene, benzoxazole, benzobisoxazole, benzothiazole, benzimidazole , benzothiophene, quinoline, isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine, indolizine.

As used herein, a "biodegradable group" is a group that helps to accelerate the metabolism of lipids in mammalian subjects. The biodegradable group may be selected from, but is not limited to, the group consisting of: -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R') C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR' )O-, -S(O) _2- , the group consisting of aryl and heteroaryl. As used herein, "aryl" is an optionally substituted carbocyclic group containing one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl. As used herein, "heteroaryl" is an optionally substituted heterocyclic group containing one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, and thiazolyl. Both aryl and heteroaryl groups can be optionally substituted. For example, M and M' can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole and thiazole. In the formulae herein, M and M' can be independently selected from the above list of biodegradable groups. Unless otherwise specified, an aryl or heteroaryl group described herein refers to both unsubstituted and substituted groups, ie, an optionally substituted aryl or heteroaryl group.

Unless otherwise specified, alkyl, alkenyl, and cyclyl groups (eg, carbocyclyl and heterocyclyl) can be optionally substituted. Optional substituents may be selected from, but are not limited to, the group consisting of halogen atoms (eg, chloro, bromo, fluoro, or iodo), carboxylic acids (eg, -C(O)OH), alcohols ( For example, hydroxyl, -OH), ester (eg, -C(O)OR or -OC(O)R), aldehyde (eg, -C(O)H), carbonyl (eg, -C(O)R or represented by C=O), halogenated acyl groups (eg, -C(O)X, where X is a halide selected from bromide, fluoride, chloride, and iodide), carbonates (eg, -OC(O) OR), alkoxy (eg, -OR), acetal (eg, _-C (OR)2R"", where each OR is the same or a different alkoxy and R"" is an alkyl or alkenyl group ), phosphoric acid (eg, P(O) ₄ ^3- ), thio (eg, -SH), sulfoxide (eg, -S(O)R), sulfinic acid (eg, -S(O)OH) ), sulfonic acid (eg, -S(O) ₂ OH), thioaldehyde (eg, -C(S)H), sulfuric acid (eg, S(O) ₄ ^2- ), sulfonyl (eg, -S( O) _2- ), amide (eg, _-C (O)NR2 or -N(R)C(O)R), azide (eg, _-N3 ), nitro (eg, _-NO2 ) , cyano (eg, -CN), isocyano (eg, -NC), acyloxy (eg, -OC(O)R), amino (eg, -NR2, -NRH, or _{-NH2 )} , amino Formyl (eg, -OC(O)NR2, -OC(O)NRH, or -OC(O) _{NH2 )} , sulfonamide (eg, -S(O) _2NR2 , -S(O _{) 2NRH} , -S(O) ₂ NH ₂ , -N(R)S(O) ₂ R, -N(H)S(O) ₂ R, -N(R)S(O) ₂ H or -N(H )S(O ₎ 2H), alkyl, alkenyl, and cyclyl (eg, carbocyclyl or heterocyclyl). In any of the foregoing, R is alkyl or alkenyl as defined herein. In some embodiments, the substituents themselves may be further substituted with, for example, one, two, three, four, five or six substituents as defined herein. For example, a _C1-6 alkyl group can be further substituted with one, two, three, four, five or six substituents as described herein.

Nitrogen containing compounds of the present disclosure can be converted to N-oxides by treatment with oxidizing agents (eg, 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxide) to give other compounds of the present disclosure. Accordingly, all shown and claimed nitrogen-containing compounds are considered to include the compounds shown and their N-oxide derivatives (which may be referred to as N→O or N ⁺ -O ^- ) when valence and structure permit. Furthermore, in other instances, nitrogens in the compounds of the present disclosure can be converted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxy compounds can be prepared by oxidizing the parent amine with an oxidizing agent such as m-CPBA. All shown and claimed nitrogen-containing compounds are also considered to cover the compounds shown and their N-hydroxyl (ie N-OH) and N-alkoxy (ie N-OR, where R is N-OH) and N-alkoxy (ie N-OR) compounds when valence and structure permit Substituted or unsubstituted C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, 3-14 membered carbocycle or 3-14 membered heterocycle) derivatives.

About, approximately: As used herein, the terms "approximately" and "about" when applied to the value or values concerned refer to a value similar to the referenced value. In certain embodiments, unless otherwise specified or otherwise apparent from context, the terms "about" or "about" refer to 25%, 20%, 19% in either direction (greater or less than) of the reference value , 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2 A range of values in the range of %, 1% or less (except where this value would exceed 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean +/- 10% of the stated value. For example, a nanoparticle composition comprising a lipid component with about 40% of a given compound may comprise 30-50% of the compound.

As used herein, the term "compound" is intended to include all isomers and isotopes having the depicted structure. "Isotopes" refer to atoms with the same atomic number but different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Additionally, the compounds, salts or complexes of the present disclosure can be prepared in combination with solvents or water molecules to form solvates and hydrates by conventional methods.

As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical link. Methods of contacting cells with external entities in vivo and ex vivo are well known in the field of biology. For example, contacting a nanoparticle composition with mammalian cells in a mammal can be by different routes of administration (eg, intravenous, intramuscular, intradermal, and subcutaneous) and can include varying amounts of the nanoparticle composition (eg, Empty LNP or Loaded LNP). Additionally, the nanoparticle composition can be contacted with more than one mammalian cell.

As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a therapeutic and/or prophylactic agent to a subject can include administering to the subject a nanoparticle composition comprising the therapeutic and/or prophylactic agent (eg, via intravenous, intramuscular, intradermal or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cells can include contacting one or more cells with the nanoparticle composition.

As used herein, the term "enhanced delivery" means that the level of therapeutic and/or prophylactic (eg, MC3, KC2, or DLinDMA) delivered by a nanoparticle compared to the level of therapeutic and/or prophylactic (eg, MC3, KC2, or DLinDMA) delivered to a target tissue of interest by a control nanoparticle More (eg, at least 1.5 times more, at least 2 times more, at least 3 times more, at least 4 times more, at least 5 times more) therapeutic and/or prophylactic agents delivered to the target tissue of interest (eg, mammalian liver) times, at least 6 times more, at least 7 times more, at least 8 times more, at least 9 times more, at least 10 times more). The level of nanoparticles delivered to a particular tissue can be determined by comparing the amount of protein produced in the tissue to the weight of the tissue, the amount of therapeutic and/or prophylactic agent in the tissue to the weight of the tissue, and The amount of protein produced in the tissue is measured by comparing the amount of total protein in the tissue or by comparing the amount of therapeutic and/or prophylactic agent in the tissue with the amount of total therapeutic and/or preventive agent in the tissue . It will be appreciated that it is not necessary to measure the enhanced delivery of nanoparticles to target tissues in the subject being treated, it can be measured in surrogates such as animal models (eg, rat models). In certain embodiments, comprising according to formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I- Ya) Nanoparticles of Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) The compositions have substantially the same level of delivery enhancement regardless of the route of administration. For example, certain compounds disclosed herein exhibit similar delivery enhancements when used for intravenous or intramuscular delivery of therapeutic and/or prophylactic agents. In other embodiments, certain compounds disclosed herein exhibit higher levels of delivery enhancement when used for intramuscular delivery of therapeutic and/or prophylactic agents than when administered intravenously.

As used herein, the term "specific delivery/specifically deliver/specifically delivering" means delivery by nanoparticles to target tissues of interest (eg, mammalian spleen) as compared to off-target tissues (eg, mammalian spleen). animal liver) more (eg, at least 1.5 times more, at least 2 times more, at least 3 times more, at least 4 times more, at least 5 times more, at least 6 times more, at least 7 times more , at least 8 times more, at least 9 times more, at least 10 times more). The level of nanoparticles delivered to a particular tissue can be determined by comparing the amount of protein produced in the tissue to the weight of the tissue, the amount of therapeutic and/or prophylactic agent in the tissue to the weight of the tissue, and The amount of protein produced in the tissue is measured by comparing the amount of total protein in the tissue or by comparing the amount of therapeutic and/or prophylactic agent in the tissue with the amount of total therapeutic and/or preventive agent in the tissue . For example, for renal vascular targeting, if the therapeutic and/or prophylactic agent is administered systemically, the therapeutic and/or prophylactic agent delivered to the kidney will be 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 15-fold or 20-fold more 1 g of tissue, the therapeutic and/or prophylactic agent is specifically delivered to the mammalian kidney compared to the liver and spleen. It will be appreciated that the ability of nanoparticles to specifically deliver to target tissues does not have to be determined in the subject being treated, but can be determined in surrogate models such as animal models (eg, rat models).

As used herein, "encapsulation efficiency" refers to the amount of therapeutic and/or prophylactic agent that becomes part of a nanoparticle composition versus the initial total amount of therapeutic and/or prophylactic agent used to prepare the nanoparticle composition ratio. For example, if 97 mg of the therapeutic and/or prophylactic agent is encapsulated in the nanoparticle composition out of a total of 100 mg of therapeutic and/or prophylactic agent initially provided to the composition, an encapsulation efficiency of 97% can be given. As used herein, "encapsulating" may refer to encapsulating, sealing, enclosing, or wrapping in whole, in part, or in part.

As used herein, "encapsulated," "encapsulated," "loaded," and "associated with" can refer to fully, mostly, or partially encapsulating, sealing, surrounding, or wrapping. As used herein, "encapsulation" or "association" can refer to the process of confining a single nucleic acid molecule within a nanoparticle and/or establishing a physicochemical relationship between a single nucleic acid molecule and a nanoparticle. As used herein, "empty nanoparticles" can refer to nanoparticles that are substantially free of therapeutic or prophylactic agents. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleic acid. As used herein, "empty nanoparticles" or "empty lipid nanoparticles" can refer to nanoparticles that are substantially free of nucleotides or polypeptides. As used herein, "empty nanoparticle" or "empty lipid nanoparticle" may refer to a nanoparticle consisting essentially of only lipid components. As used herein, a "loaded nanoparticle" or "loaded lipid nanoparticle" (also referred to as "intact nanoparticle" or "intact lipid nanoparticle") may refer to a nanoparticle comprising an empty nanoparticle and a therapeutic or prophylactic component Nanoparticles. As used herein, "loaded nanoparticle" or "loaded lipid nanoparticle" (also referred to as "intact nanoparticle" or "intact lipid nanoparticle") may refer to a nanoparticle comprising an empty nanoparticle and a nucleotide or polypeptide component Nanoparticles. As used herein, "loaded nanoparticle" or "loaded lipid nanoparticle" (also referred to as "intact nanoparticle" or "intact lipid nanoparticle") may refer to a nanoparticle comprising empty nanoparticles and nucleic acid components.

As used herein, "expression" of a nucleic acid sequence refers to translation of mRNA into polypeptides or proteins and/or post-translational modifications of polypeptides or proteins.

As used herein, the term "in vitro" refers to taking place in an artificial environment (eg, in a test tube or reaction vessel, in a cell culture, a Petri dish, etc.) rather than in an organism (eg, an animal, plant or events occurring in microorganisms).

As used herein, the term "in vivo" refers to events that occur within an organism, such as an animal, plant, or microorganism, or cells or tissues thereof.

As used herein, the term "ex vivo" refers to an event that occurs outside an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered relative to the natural (eg, in vivo) environment.

As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may contain one or more chiral centers and/or double bonds, and thus take the form of stereoisomers such as double bond isomers (ie, geometric E/Z isomers) or diastereomers (eg, para- Exist as enantiomers (ie, (+) or (-)) or cis/trans isomers. The present disclosure encompasses any and all isomers of the compounds described herein, including stereoisomerically pure forms (eg, geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomerically pure Constituents and stereoisomer mixtures, such as racemates. Enantiomeric and stereoisomeric mixtures of compounds and the manner in which they are resolved into their constituent enantiomers or stereoisomers are well known.

A "tautomer" is one of two or more structural isomers that exist in equilibrium and are readily convertible from one isomeric form to another. This transformation induces formal migration of hydrogen atoms accompanied by transformation of adjacent conjugated double bonds. Tautomers exist in solution as mixtures of tautomeric groups. In solutions where tautomerism can occur, a chemical equilibrium of tautomers will be reached. The exact ratio of tautomers depends on several factors, including temperature, solvent and pH. The concept of tautomers that can be interconverted by tautomerization is called tautomerism.

Of the various types of tautomerism that may exist, two are frequently observed. In keto-enol tautomerism, shifts of electrons and hydrogen atoms occur simultaneously. Ring-chain tautomerism is caused by the reaction of an aldehyde group (-CHO) in a sugar chain molecule with a hydroxyl group (-OH) in the same molecule to give the molecule a cyclic (ring) form as exhibited by glucose of.

Common tautomeric pairs are: keto-enol, amide-nitrile, lactam-lactam, amide-imide in heterocycles (eg in nucleobases such as guanine, thymine and cytosine) Amino acid tautomerism, imine-enamine and enamine-enamine. Examples of tautomerism in disubstituted guanidines are shown below.

It will be appreciated that the compounds of the present disclosure may be depicted with different tautomers. It is also to be understood that when compounds have tautomeric forms, all tautomeric forms are intended to be included within the scope of this disclosure and that the naming of the compounds does not exclude any tautomeric forms.

As used herein, a "lipid component" is a component of a nanoparticle composition comprising one or more lipids. For example, the lipid component may comprise one or more cationic/ionizable, PEGylated, structural or other lipids, such as phospholipids.

As used herein, a "linker" is a moiety that connects two moieties, eg, a link between two nucleosides of a capping species. The linker may contain one or more groups including, but not limited to, phosphate groups (eg, phosphoric acid, boronophosphoric acid, thiophosphoric acid, selenophosphoric acid, and phosphonic acid), alkyl, amidate, or glycerol. For example, the two nucleosides of a cap analog can be linked at their 5' position by a triphosphate group or by a chain comprising two phosphate moieties and one boronophosphate moiety.

As used herein, "administration method" may include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering the composition to a subject. The method of administration can be selected for targeted delivery (eg, specific delivery) to a given area or system of the body.

As used herein, "modified" means non-natural. For example, the RNA can be modified RNA. That is, the RNA can comprise one or more non-naturally occurring nucleobases, nucleosides, nucleotides or linkers. A "modified" substance may also be referred to herein as an "altered" substance. Substances may be modified or altered chemically, structurally or functionally. For example, modified nucleobase species may contain one or more non-naturally occurring substitutions.

As used herein, "N:P ratio" is the ratio of ionizable (in the physiological pH range) nitrogen atoms in lipids to phosphate groups in RNA, eg, in a nanoparticle composition comprising a lipid component and RNA The molar ratio of.

As used herein, a "nanoparticle composition" is a composition comprising one or more lipids. Nanoparticle compositions are typically micron-scale or smaller in size and may contain lipid bilayers. Nanoparticle compositions encompass lipid nanoparticles (LNPs), liposomes (eg, lipid vesicles), and liposome complexes. For example, the nanoparticle composition can be a liposome with a lipid bilayer having a diameter of 500 nm or less.

As used herein, "naturally occurring" means existing in nature without artificial assistance.

As used herein, "patient" refers to a subject who may seek or be in need of, is in need of, is receiving, will receive, or is under the care of a trained professional for a particular disease or condition .

As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid comprising a polyethylene glycol component.

The phrase "pharmaceutically acceptable" is used herein to mean, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications, and with reasonable Compounds, materials, compositions and/or dosage forms that are consistent with the benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than a compound described herein that has the property of being substantially non-toxic and non-inflammatory in a patient (eg, capable of rendering an active compound suspending, complexing or dissolving vehicles). Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), softeners, emulsifiers, fillers (diluents), film formers Or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crospovidone, citric acid , crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine Acid, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate , Shellac, Silicon Dioxide, Sodium Carboxymethyl Cellulose, Sodium Citrate, Sodium Starch Glycolate, Sorbitol, Starch (Corn), Stearic Acid, Sucrose, Talc, Titanium Dioxide, Vitamin A, Vitamin E ( alpha-tocopherol), vitamin C, xylitol, and others disclosed herein.

In the present specification, the structural formula of a compound presents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometric isomers, asymmetric carbon-based optical isomers, stereoisomers isomers, tautomers, etc., it should be understood that not all isomers have the same level of activity. Furthermore, the compounds represented by the formulae may exist in crystalline polymorphs. It should be noted that any crystal form, mixture of crystal forms, or anhydrides or hydrates thereof are included within the scope of the present disclosure.

The term "crystalline polymorph", "polymorph" or "crystalline form" means a crystal structure in which a compound (or a salt or solvate thereof) can crystallize into different crystal packing arrangements, all of which have the same elemental composition . Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may allow one crystal form to prevail. Crystalline polymorphs of the compounds can be prepared by crystallization under various conditions.

The compositions may also contain salts of one or more compounds. The salt can be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is obtained by converting an existing acid or base moiety into its salt form (eg, by concatenating the free base group with a suitable organic compound) acid reaction). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate , camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, lauryl sulfate, ethanesulfonate, fumarate, glucoheptanoate ( glucoheptonate), glycerophosphate, hemisulfate, heptanoate, caproate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate , laurate, lauryl sulfate, malate, maleate, malonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate , palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, Stearates, succinates, sulfates, tartrates, thiocyanates, tosylates, undecanoates, valerates, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, diethylammonium Methylamine, trimethylamine, triethylamine, ethylamine, etc. Pharmaceutically acceptable salts of the present disclosure include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally , preferably non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile.

As used herein, a "phospholipid" is a lipid comprising a phosphate moiety and one or more carbon chains such as unsaturated fatty acid chains. Phospholipids may contain one or more multiple (eg, double or triple) bonds (eg, one or more unsaturations). Certain phospholipids can aid in fusion with the membrane. For example, cationic phospholipids can interact with one or more negatively charged phospholipids of membranes (eg, cell membranes or intracellular membranes). Fusion of the phospholipid to the membrane can allow one or more components of the lipid-containing composition to pass through the membrane, allowing, for example, delivery of the one or more components to a cell.

As used herein, a "polydispersity index" or "PDI" is a ratio that describes the homogeneity of the particle size distribution of a system. Smaller values, eg less than 0.3, indicate a narrower particle size distribution.

As used herein, the term "polypeptide" or "polypeptide of interest" refers to a polymer of amino acid residues, usually joined by peptide bonds, which may be naturally occurring (eg, isolated or purified) or synthetically produced. The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may contain modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as with labeling components). conjugation). Also included in the definition are, for example, amino acids containing one or more analogs of amino acids (including, for example, unnatural amino acids such as homocysteine, ornithine, para-acetylphenylalanine, D-amino acids, and creatine) and the present Other modified polypeptides known in the art. As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologues, paralogues, fragments of the foregoing and other equivalents, variants and analogs. Polypeptides may be monomers or may be multimolecular complexes such as dimers, trimers or tetramers. They may also include single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" may be less than or equal to 50 amino acids long, eg, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

As used herein, "RNA" refers to ribonucleic acid that may or may not occur naturally. For example, RNA can comprise modified and/or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may contain cap structures, chain terminating nucleosides, stem loops, polyA sequences and/or polyadenylation signals. The RNA can have a nucleotide sequence encoding the polypeptide of interest.

As used herein, "DNA" refers to deoxyribonucleic acid that may or may not occur naturally. For example, the DNA can be a synthetic molecule, such as a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. As used herein, a "recombinant DNA molecule" refers to a DNA molecule that does not exist as a natural product but is produced using molecular biology techniques.

As used herein, a "single unit dose" is the dose of any therapeutic agent administered in one dose/one time/single route/single point of contact, ie, a single administration event.

As used herein, "fractionated dose" is the division of a single unit dose or total daily dose into two or more doses.

As used herein, a "total daily dose" is the amount administered or prescribed over a 24 hour period. The total daily dose can be administered as a single unit dose.

As used herein, "size" or "average size" in the context of lipid nanoparticles (eg, empty LNPs or loaded LNPs) refers to the average diameter of the nanoparticle composition.

As used herein, the term "subject" or "patient" refers to any organism to which a composition according to the present disclosure can be administered, eg, for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

As used herein, a "targeted cell" refers to any one or more cells of interest. These cells can be found in vitro, in vivo, in situ, or in tissues or organs of an organism. The organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.

As used herein, "target tissue" refers to any one or more tissue types of interest in which delivery of a therapeutic and/or prophylactic agent will elicit a desired biological and/or pharmacological effect. Examples of target tissues of interest include designated tissues, organs, and systems, or groups thereof. In certain applications, the target tissue may be the kidney, lung, spleen, vascular endothelium in a blood vessel (eg, intracoronary or intrafemoral), or tumor tissue (eg, by intratumoral injection). As used herein, "off-target tissue" refers to any tissue type or types in which expression of the encoded protein does not result in the desired biological and/or pharmacological effect. In certain applications, off-target tissues may include liver and spleen.

The term "therapeutic agent" or "prophylactic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. Therapeutic agents are also referred to as "active agents" or "active agents." Such agents include, but are not limited to, cytotoxins, radioactive ions, chemotherapeutics, small molecule drugs, proteins, and nucleic acids.

As used herein, the term "therapeutically effective amount" means sufficient to treat, ameliorate the symptoms of, an infection, disease, disorder and/or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder and/or condition , the amount of an agent (eg, nucleic acid, drug, composition, therapeutic, diagnostic, prophylactic, etc.) delivered to diagnose, prevent, and/or delay its onset.

As used herein, "transfection" refers to the introduction of a substance (eg, RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.

As used herein, the term "treating" refers to partially or fully alleviating, ameliorating, ameliorating, alleviating, delaying onset, inhibiting progression, reducing one or more symptoms or characteristics of a particular infection, disease, disorder and/or condition its severity and/or reduce its incidence. For example, "treating" cancer can refer to inhibiting the survival, growth and/or spread of a tumor. Subjects who do not exhibit signs of the disease, disorder and/or condition and/or only exhibit disease, disorder and/or Treatment is administered to subjects with early signs of the condition.

As used herein, "zeta potential" is, for example, the zeta potential of lipids in a nanoparticle composition.

Nanoparticle composition

The present disclosure also features the inclusion of formulas (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y) as described herein , (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) Compounds of lipid nanoparticles.

In some embodiments, the nanoparticle composition has a largest dimension of 1 μm or less when measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method (eg, 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm or shorter). Nanoparticle compositions include, for example, lipid nanoparticles (LNPs; eg, empty LNPs or loaded LNPs), liposomes, lipid vesicles, and liposome complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In certain embodiments, the nanoparticle composition includes two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and/or cross-linked to each other. The lipid bilayer may contain one or more ligands, proteins or channels.

The nanoparticle composition comprises a lipid component comprising at least one according to formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb). For example, the lipid component of the nanoparticle composition may comprise one or more compounds of Table 1. The nanoparticle composition may also contain a variety of other components. For example, lipid components of nanoparticle compositions other than those according to formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y) , (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) In addition to lipids, one or more other lipids may also be included.

Cationic/Ionizable Lipids

Nanoparticles (eg, empty LNP or loaded LNP) except according to formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y) , (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) In addition to lipids, one or more cationic and/or ionizable lipids (eg, lipids that may be positively or partially positively charged at physiological pH) may be included. Cationic and/or ionizable lipids may be selected from the non-limiting group consisting of: 3-(di-dodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(di-dodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethylamine (KL22), 14,25 -Di-tridecyl-15,18,21,24-tetraaza-trioctadecane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin -DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino) Heptadeca-6,9,28,31-tetraen-19-yl butyrate (DLin-MC3-DMA), 2,2-dilinole-4-(2-dimethylaminoethyl) )-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8 -[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12- Dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octane yl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA( 2R)) and (2S)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[ (9Z,12Z)-Octadec-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA(2S)). In addition to these, cationic lipids may also be lipids containing cyclic amine groups.

structured lipids

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can comprise one or more structured lipids. The structural lipid may be selected from, but is not limited to, the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomato Tomatidine, tomatine, ursolic acid, alpha-tocopherol and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. In some embodiments, the structured lipid is:

Phospholipids

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) may comprise one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can assemble into one or more lipid bilayers. In general, a phospholipid may comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid can be a lipid according to formula (IV):

wherein R _p represents a phospholipid moiety and ^RA and ^RB represent fatty acid moieties with or without unsaturation, which may be the same or different. The phospholipid moiety can be selected from the non-limiting group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid , phytanic acid, arachidonic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. It is also contemplated to include non-natural materials with modified and substituted natural materials, including branching, oxidation, cyclization, and alkynes. For example, phospholipids can be functionalized with one or more alkynes (eg, alkenyl groups in which one or more double bonds are replaced by triple bonds) or crosslinked with one or more alkynes. Under appropriate reaction conditions, alkynyl groups may undergo copper-catalyzed cycloaddition reactions when exposed to azides. Such reactions can be used to functionalize the lipid bilayer of nanoparticles (eg, empty or loaded LNPs) to facilitate membrane penetration or cell recognition, or to conjugate nanoparticles (eg, empty or loaded LNPs) to useful groups. Such as targeting moieties or imaging moieties (eg dyes).

Phospholipids useful in these compositions and methods can be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl -sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine Base (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2 - Di-undecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- Octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1- Cetyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl -sn-glycero-3-phosphocholine, 1,2-di-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- Phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenic acid Acyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di-docosahexaenoyl-sn-glycero-3- Phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine ( POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oil Acylphosphatidylcholines, lysophosphatidylcholines, lysophosphatidylethanolamines (LPE) and mixtures thereof. In some embodiments, the lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprise DSPC. In certain embodiments, the lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprise DOPE. In some embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprise both DSPC and DOPE.

PEG lipids

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can comprise one or more PEG or PEG-modified lipids. These substances may alternatively be referred to as PEGylated lipids. PEG lipids are lipids modified with polyethylene glycol. PEG lipids can be selected from the non-limiting group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (PEG-CER), PEG-modified dialkylamine, PEG-modified dialkylamine Acylglycerols (PEG-DEG), PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

In certain embodiments, the PEG lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol , PEG-modified dialkylglycerol.

In certain embodiments, the PEG lipid is selected from the group consisting of 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl -sn-glycerol-3-phosphoethanolamine-N-[amino(polyethylene glycol)](PEG-DSPE), PEG-distearylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl Acyl, PEG-distearyl, PEG-diacylglycinamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE) or PEG-1,2-dimyristyloxypropyl -3-amine (PEG-c-DMA). For example, in some embodiments, the PEG lipid is PEG-DMG.

In certain embodiments, the PEG lipid is a compound of formula (PL-I):

or a salt thereof, where:

R ^3PL1 is –OR ^OPL1 ;

R ^OPL1 is hydrogen, an optionally substituted alkyl or oxygen protecting group;

r ^PL1 is an integer between 1 and 100, inclusive;

L ¹ is an optionally substituted C _1-10 alkylene group, wherein at least one methylene group in the optionally substituted C _1-10 alkylene group is independently replaced by: optionally substituted carbocyclylene, any optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R ^NPL1 ), S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R ^NPL1 ), NR ^NPL1 C(O)O or NR ^NPL1 C(O)N( R ^NPL1 );

D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

m ^PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A has the following formula:

Each instance of L is ^{independently} a bond or an optionally substituted _C1-6 alkylene where one methylene unit in the optionally substituted _C1-6 alkylene is optionally replaced with: O , N(R ^NPL1 ), S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), C(O)O, OC(O), OC(O)O, OC( O)N(R ^NPL1 ), NR ^NPL1 C(O)O or NR ^NPL1 C(O)N(R ^NPL1 );

Each instance of R ^2SL is independently optionally substituted C _1-30 alkyl, optionally substituted C _1-30 alkenyl, or optionally substituted C _1-30 alkynyl; optionally one of wherein R ^2SL or more methylene units are independently replaced with: optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N( R ^NPL1 ), O, S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), NR ^NPL1 C(O)N(R ^NPL1 ), C(O)O, OC( O), OC(O)O, OC(O)N(R ^NPL1 ), NR ^NPL1 C(O)O, C(O)S, SC(O), C(=NR ^NPL1 ), C(=NR ^NPL1 )N(R ^NPL1 ), NR ^NPL1 C(=NR ^NPL1 ), NR ^NPL1 C(=NR ^NPL1 )N(R ^NPL1 ), C(S), C(S)N(R ^NPL1 ), NR ^NPL1 C(S ), NR ^NPL1 C(S)N(R ^NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O) ₂ , S(O) ₂ O, OS (O) ₂ O, N(R ^NPL1 )S(O), S(O)N(R ^NPL1 ), N(R ^NPL1 )S(O)N(R ^NPL1 ), OS(O)N(R ^NPL1 ) , N(R ^NPL1 )S(O)O, S(O) ₂ , N(R ^NPL1 )S(O) ₂ , S(O) ₂ N(R ^NPL1 ), N(R ^NPL1 )S(O) ₂ N(R ^NPL1 ), OS(O) ₂ N(R ^NPL1 ) or N(R ^NPL1 )S(O) ₂ O;

Each instance of R ^NPL1 is independently a hydrogen, optionally substituted alkyl or nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

^pSL is 1 or 2.

In certain embodiments, the PEG lipid is a compound of formula (PL-I-OH):

或其盐。

or its salt.

In certain embodiments, the PEG lipid is a compound of formula (PL-II-OH):

或其盐或异构体，其中：

or a salt or isomer thereof, wherein:

R ^3PEG is -OR ^O ;

R ^O is a hydrogen, C _1-6 alkyl or oxygen protecting group;

r ^PEG is an integer between 1 and 100;

R ^5PEG is C _10-40 alkyl, C _10-40 alkenyl, or C _10-40 alkynyl; and optionally one or more methylene groups of R ^5PEG are independently replaced with: C _3-10 carbonylene Cyclic, 4- to 10-membered heterocyclylene, C _6-10 -membered arylene, 4- to 10-membered heteroarylene, –N(R ^NPEG )–, –O–, –S–, –C(O) –, –C(O)N(R ^NPEG )–, –NR ^NPEG C(O)–, –NR ^NPEG C(O)N(R ^NPEG )–, –C(O)O–, –OC(O) –, –OC(O)O–, –OC(O)N(R ^NPEG )–, –NR ^NPEG C(O)O–, –C(O)S–, –SC(O)–, –C( =NR ^NPEG )–, –C(=NR ^NPEG )N(R ^NPEG )–, –NR ^NPEG C(=NR ^NPEG )–, –NR ^NPEG C(=NR ^NPEG )N(R ^NPEG )–, –C( S)–, –C(S)N(R ^NPEG )–, –NR ^NPEG C(S)–, –NR ^NPEG C(S)N(R ^NPEG )–, –S(O)–, –OS(O )–, –S(O)O–, –OS(O)O–, –OS(O) ₂ –, –S(O) ₂ O–, –OS(O) ₂ O–, –N(R ^NPEG )S(O)–, –S(O)N(R ^NPEG )–, –N(R ^NPEG )S(O)N(R ^NPEG )–, –OS(O)N(R ^NPEG )–, –N (R ^NPEG )S(O)O–, –S(O) ₂ –, –N(R ^NPEG )S(O) ₂ –, –S(O) ₂ N(R ^NPEG )–, –N(R ^NPEG )S(O) ₂ N(R ^NPEG )–, –OS(O) ₂ N(R ^NPEG )– or –N(R ^NPEG )S(O) ₂ O–; and

Each instance of R ^NPEG is independently a hydrogen, _C1-6 alkyl or nitrogen protecting group.

In certain embodiments, in the PEG lipid of formula (PL-II-OH), r is an integer between 40 and 50. For example, r is selected from the group consisting of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. For example, r is 45.

In certain embodiments, in the PEG lipid of formula (PL-II ^- OH), R5 is _C17 alkyl.

In certain embodiments, the PEG lipid is a compound of formula (PL-II):

其中r^PEG是介于1与100之间的整数。

where ^rPEG is an integer between 1 and 100.

In certain embodiments, the PEG lipid is a compound of formula (PEG-1):

In certain embodiments, the PEG lipid is a compound of formula (PL-III):

或其盐或异构体，其中s^PL1是介于1与100之间的整数。

or a salt or isomer thereof, wherein s ^PL1 is an integer between 1 and 100.

In certain embodiments, the PEG lipid is a compound of the formula:

In certain embodiments, nanoparticle formulations incorporate formula (PL-I), PL-I-OH), (PL-II), (PL-II-OH), (PL-III), PEG _2k A lipid of one of DMG or PEG-1, which can improve the pharmacokinetics and/or biodistribution of lipid nanoparticle formulations. For example, incorporation of lipids of one of the formulae (PL-II-OH), (PL-IIa-OH), (PL-II) or PEG-1 in nanoparticle formulations can reduce the accelerated blood clearance (ABC) effect .

adjuvant

In some embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising one or more lipids described herein may also comprise one or more adjuvants, eg, glucopyranosyl lipids Adjuvants (GLA), CpG oligodeoxynucleotides (eg, class A or class B), poly(I:C), aluminum hydroxide, and Pam3CSK4.

therapeutic agent

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can contain one or more therapeutic and/or prophylactic agents. The present disclosure features methods of delivering therapeutic and/or prophylactic agents to mammalian cells or organs, producing polypeptides of interest in mammalian cells, and treating a disease or disorder in a mammal in need thereof, including administering to a mammalian The animals are administered lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising therapeutic and/or prophylactic agents and/or contact mammalian cells with the lipid nanoparticles.

Therapeutic and/or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic and/or prophylactic agents can be substances that, once delivered to the cell or organ, cause a desired change in the cell, organ, or other body tissue or system. Such substances can be used to treat one or more diseases, disorders or conditions. In some embodiments, the therapeutic and/or prophylactic agent is a small molecule drug that can be used to treat a particular disease, disorder or condition.

In some embodiments, the therapeutic and/or prophylactic agent is a vaccine, a compound that elicits an immune response (eg, a polynucleotide or nucleic acid molecule encoding a protein or polypeptide or peptide, or a protein or polypeptide, or a protein) and/or another A therapeutic and/or preventive agent. Vaccines comprise compounds and preparations capable of providing immunity against one or more conditions associated with infectious disease, and may comprise mRNA encoding infectious disease-derived antigens and/or epitopes. Vaccines may also include compounds and preparations that direct an immune response against cancer cells, and may include mRNA encoding tumor cell-derived antigens, epitopes, and/or neo-epitopes. In some embodiments, vaccines and/or compounds capable of eliciting an immune response are administered intramuscularly by the compositions of the present disclosure.

In other embodiments, the therapeutic and/or prophylactic agent is a protein, eg, a protein required to supplement or replace the naturally occurring protein of interest. Such proteins or polypeptides may be naturally occurring, or may be modified using methods known in the art, eg, to increase half-life. Exemplary proteins are intracellular, transmembrane, or secreted.

Polynucleotides and Nucleic Acids

In some embodiments, the therapeutic agent is an agent that enhances (ie, increases, stimulates, upregulates) protein expression. Non-limiting examples of the types of therapeutic agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR/Cas9 technology, ssDNA, and DNA (eg, expression vectors). Agents that upregulate protein expression can upregulate the expression of naturally occurring or non-naturally occurring proteins (eg, chimeric proteins that have been modified to increase half-life, or chimeric proteins that contain desired amino acid changes). Exemplary proteins include intracellular, transmembrane or secreted proteins, peptides or polypeptides.

In some embodiments, the therapeutic agent is a DNA therapeutic agent. A DNA molecule can be double-stranded DNA, single-stranded DNA (ssDNA) or a molecule of partially double-stranded DNA, ie a molecule having a double-stranded portion and a single-stranded portion. In some cases, the DNA molecule is triple-stranded or partially triple-stranded, ie, has a triple-stranded portion and a double-stranded portion. The DNA molecule can be a circular DNA molecule or a linear DNA molecule.

A DNA therapeutic can be a DNA molecule capable of transferring a gene into a cell, eg, a DNA molecule that encodes and can express a transcript. In other embodiments, the DNA molecule can be a synthetic molecule, eg, a synthetic DNA molecule produced in vitro. In some embodiments, the DNA molecule is a recombinant molecule. Non-limiting exemplary DNA therapeutics include plasmid expression vectors and viral expression vectors.

The DNA therapeutics described herein, eg, DNA vectors, can include a number of different features. DNA therapeutics, such as DNA vectors, described herein, can include non-coding DNA sequences. For example, a DNA sequence can comprise at least one gene regulatory element, such as a promoter, enhancer, termination element, polyadenylation signal element, splicing signal element, and the like. In some embodiments, the non-coding DNA sequence is an intron. In some embodiments, the non-coding DNA sequence is a transposon. In some embodiments, the DNA sequences described herein may have non-coding DNA sequences operably linked to a transcriptionally active gene. In other embodiments, the DNA sequences described herein may have non-coding DNA sequences that are not linked to genes, ie, the non-coding DNA does not regulate the genes on the DNA sequences.

In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and/or prophylactic agents are nucleic acids. In some embodiments, the one or more therapeutic and/or prophylactic agents are selected from the group consisting of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

For example, in some embodiments, when the therapeutic and/or prophylactic agent is DNA, the DNA is selected from the group consisting of double-stranded DNA, single-stranded DNA (ssDNA), partially double-stranded DNA, triple-stranded DNA, and partially triple-stranded DNA group. In some embodiments, the DNA is selected from the group consisting of circular DNA, linear DNA, and mixtures thereof.

In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and/or prophylactic agents are selected from the group consisting of plasmid expression vectors, viral expression vectors, and mixtures thereof.

For example, in some embodiments, when the therapeutic and/or prophylactic agent is RNA, the RNA is selected from the group consisting of single-stranded RNA, double-stranded RNA (dsRNA), partially double-stranded RNA, and mixtures thereof. In some embodiments, the RNA is selected from the group consisting of circular RNA, linear RNA, and mixtures thereof.

For example, in some embodiments, when the therapeutic and/or prophylactic agent is RNA, the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule, MicroRNA (miRNA), antagomir, antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), locked nucleic acid (LNA) and CRISPR/Cas9 technologies and their mixture.

For example, in some embodiments, when the therapeutic and/or prophylactic agent is RNA, the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer - Substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

In some embodiments, the one or more therapeutic and/or prophylactic agents is mRNA. In some embodiments, the one or more therapeutic and/or prophylactic agents are modified mRNA (mmRNA).

In some embodiments, the one or more therapeutic and/or prophylactic agents are mRNAs that incorporate a microRNA binding site (miR binding site). Furthermore, in some embodiments, the mRNA comprises one or more of a stem-loop, chain terminating nucleoside, polyA sequence, polyadenylation signal, and/or a 5' cap structure.

mRNA can be naturally or non-naturally occurring mRNA. As described below, mRNA may contain one or more modified nucleobases, nucleosides or nucleotides, in which case it may be referred to as "modified mRNA" or "mmRNA." As used herein, a "nucleoside" is defined as containing a sugar molecule (eg, pentose or ribose) or a derivative thereof and an organic base (eg, purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleoside" nucleobases"). As used herein, a "nucleotide" is defined as a nucleoside containing a phosphate group.

An mRNA can comprise a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and/or a coding region (eg, an open reading frame). mRNA can comprise any suitable number of base pairs, including tens (eg, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100), hundreds (eg, 200, 300, 400) , 500, 600, 700, 800, or 900) or thousands (eg, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000) base pairs. Any number (eg, all, some, or none) of nucleobases, nucleosides, or nucleotides may be substituted, modified, or otherwise non-naturally occurring analogs of typical species. In certain embodiments, all specific nucleobase types can be modified. In some embodiments, all uracils or uridines are modified. When all nucleobases, nucleosides, or nucleotides are modified, eg, all uracils or uridines, mRNA may be referred to as "fully modified," eg, for uracils or uridines.

In some embodiments, an mRNA as described herein can comprise a 5' cap structure, chain termination nucleotides, optional Kozak sequences (also known as Kozak consensus sequences), stem loops, polyA sequences, and/or polyadenosine acidification signal.

A 5' cap structure or cap species is a compound comprising two nucleoside moieties joined by a linker, and can be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). The cap species may comprise one or more modified nucleosides and/or linker moieties. For example, a native mRNA cap may comprise a guanine nucleotide joined by a triphosphate linkage at its 5' position and a guanine (G) nucleotide methylated at the 7 position, such as m7G(5')ppp(5 ')G, usually written as m7GpppG. The cap material can also be an anti-reverse cap analog. A non-limiting list of possible capping species includes m7GpppG, m7Gppm7G, m73'dGpppG, m27,O3'GpppG, m27,O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG, m27, O3'GppppG and m27,O2'GppppG.

The mRNA may alternatively or additionally contain chain terminating nucleosides. For example, chain terminating nucleosides may include those nucleosides deoxygenated at the 2' and/or 3' positions of their sugar groups. Such substances may include 3' deoxyadenosine (cordycepin), 3' deoxyuridine, 3' deoxycytosine, 3' deoxyguanosine, 3' deoxythymine and 2',3' dideoxy nuclei glycosides, such as 2',3' dideoxyadenosine, 2',3' dideoxyuridine, 2',3' dideoxycytosine, 2',3' dideoxyguanosine and 2',3' dideoxy Thymine. In some embodiments, introduction of chain terminating nucleotides into the mRNA (eg, at the 3'-end) can result in stabilization of the mRNA.

The mRNA may alternatively or additionally comprise a stem-loop, such as a histone stem-loop. The stem-loop can comprise 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, a stem loop can comprise 4, 5, 6, 7 or 8 nucleotide base pairs. Stem-loops can be located in any region of the mRNA. For example, the stem-loop can be located within, before, or after an untranslated region (5' or 3' untranslated region), coding region, or polyA sequence or tail. In some embodiments, the stem-loop can affect one or more functions of the mRNA, such as translation initiation, translation efficiency, and/or transcription termination.

The mRNA may alternatively or additionally comprise a polyA sequence and/or a polyadenylation signal. The polyA sequence may consist entirely or largely of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may also contain stabilizing nucleotides or analogs. For example, the polyA sequence may contain deoxythymidine, eg, inverted (or reverse-linked) deoxythymidine (dT), as a stabilizing nucleotide or analog. Details on the use of inverted dT and other stabilizing polyA sequence modifications can be found, for example, in WO2017/049275A2, the contents of which are incorporated herein by reference. The polyA sequence may be located near the tail of the 3' untranslated region of the mRNA. In some embodiments, polyA sequences can affect nuclear export, translation and/or stability of mRNA.

The mRNA may alternatively or additionally contain a microRNA binding site. MicroRNA binding sites (or miR binding sites) can be used to regulate mRNA expression in various tissues or cell types. In an exemplary embodiment, a miR binding site is engineered into the 3'UTR sequence of the mRNA to modulate, eg, enhance the degradation of the mRNA in a cell or tissue expressing the cognate miR. Such modulation can be used to regulate or control "off-target" expression in ir mRNA, ie, expression in cells or tissues that are not desired in vivo. Details on the use of mir binding sites can be found, for example, in WO2017/062513A2, the contents of which are incorporated herein by reference.

In some embodiments, the mRNA is a bicistronic mRNA comprising a first coding region and a second coding region with an internal ribosome entry site comprising an internal ribosome entry site that allows for the initiation of internal translation between the first and second coding regions (IRES) sequences, or with intervening sequences encoding self-cleaving peptides such as the 2A peptide. IRES sequences and 2A peptides are often used to enhance the expression of multiple proteins from the same vector. Various IRES sequences are known and available in the art and can be used, including, for example, the encephalomyocarditis virus IRES.

In some embodiments, the mRNA of the present disclosure comprises one or more modified nucleobases, nucleosides or nucleotides (referred to as "modified mRNA" or "mmRNA"). In some embodiments, modified mRNAs may have useful properties compared to a reference unmodified mRNA, including enhanced stability, intracellular retention, enhanced translation, and/or lack of innate immunity to cells into which the mRNA was introduced Substantial induction of the response. Thus, the use of modified mRNA can increase the efficiency of protein production, intracellular retention of nucleic acids, and have reduced immunogenicity.

In some embodiments, the mRNA comprises one or more (eg, 1, 2, 3, or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the mRNA comprises one or more (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) different modified nucleobases, nucleosides or nucleotides. In some embodiments, the degradation of a modified mRNA in a cell into which the mRNA is introduced may be reduced relative to the corresponding unmodified mRNA.

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio Sub-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-Hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (eg, 5-iodo-uridine or 5-bromo-uridine), 3-methyl- Uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine methyl 5-oxyacetate (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxymethyl-uridine (chm5U), 5-carboxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl - Uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl yl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-aminomethyl Acylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl - Uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2- Thio-uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e. with nucleobase deoxythymine), 1-methyl - Pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl - Pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio- 1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- Pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl yl-3-(3-amino-3-carboxypropyl) pseudouridine (acp3ψ), 5-(prenylaminomethyl)uridine (inm5U), 5-(prenylaminomethyl) -2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl yl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl -2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O -Methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um) and 5-(prenylaminomethyl)-2'-O-methyl-uridine ( inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabino-uridine, 2'-F-uridine, 2'-OH-arabino-uridine, 5-(2- Carboxymethoxyvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides with modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl Acyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g. , 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- Cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudo-isocytidine, 4-thio-1-methyl-pseudo-isocytidine, 4-thio-1 -Methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebraline, 5-methyl Base-Zebraline, 5-Aza-2-Thio-Zebraline, 2-Thio-Zebraline, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl Base-cytidine, 4-methoxy-pseudo-isocytidine, 4-methoxy-1-methyl-pseudo-isocytidine, lyscytidine (k2C), α-thio-cytidine, 2'- O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2 '-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm ), 1-thio-cytidine, 2'-F-arabino-cytidine, 2'-F-cytidine, and 2'-OH-arabino-cytidine.

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with modified adenines include α-thio-adenosine, 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (eg, 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-de nitrogen-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza- 2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6- Methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-prenyl-adenosine (i6A), 2-methylthio-N6-iso Pentenyl-adenosine (ms2i6A), N6-(cis-hydroxyprenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyprenyl)adenosine ( ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine ( m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl- Adenosine (hn6A), 2-methylthio-N6-hydroxynorvaylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2 -Methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl - Adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyl Adenosine (phosphate) (Ar(p)), 2-Amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine , 2'-F-adenosine, 2'-OH-arabino-adenosine and N6-(19-amino-pentoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanines include α-thio-guanosine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylphosphoryl Rugoside (mimG), 4-desmethyl-hyosine (imG-14), isohyosine (imG2), hydrin (yW), peroxyhydrin (o2yW), hydroxyhydrin ( OhyW), undermodified hydroxybutinoside (OhyW*), 7-deaza-guanosine, braidin (Q), epoxybraidin (oQ), galactosyl-braidin (galQ), Mannosyl-braidin (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), gupurine (G+), 7-Deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine glycoside, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7 -Dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2 -Methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O -Methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1, 2'-O-Dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arabino-guanosine and 2'-F-guanosine.

In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases).

In some embodiments, the modified nucleobase is pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 2-thiouridine, 4'-thiouridine, 5-methylcytidine Pyrimidine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2- Thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine Uridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2' -O-Methyluridine. In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases). In some embodiments, the modified nucleobase is N1-methylpseudouridine (m1ψ), and the mRNA of the present disclosure is fully modified with N1-methylpseudouridine (m1ψ). In some embodiments, N1-methylpseudouridine (m1ψ) accounts for 75%-100% of the uracil in the mRNA. In some embodiments, N1-methylpseudouridine (m1ψ) accounts for 100% of the uracil in the mRNA.

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides with modified cytosines include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (eg, 5-iodo-cytidine) - cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine . In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases).

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with modified adenines include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl- Adenosine (m6A). In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases).

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanines include inosine (I), 1-methyl-inosine (m1I), hyosine (imG), methyl hyosine (mimG), 7- Deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-Methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine. In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases).

In some embodiments, the modified nucleobase is 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine or α-thio-adenosine. In some embodiments, an mRNA of the present disclosure comprises a combination of one or more of the foregoing modified nucleobases (eg, a combination of 2, 3, or 4 of the foregoing modified nucleobases).

In some embodiments, the mRNA comprises pseudouridine (ψ). In some embodiments, the mRNA comprises pseudouridine (ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ). In some embodiments, the mRNA comprises 1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2-thiouridine (s2U). In some embodiments, the mRNA comprises 2-thiouridine and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U). In some embodiments, the mRNA comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine (m5C). In some embodiments, the mRNA comprises 2'-O-methyluridine. In some embodiments, the mRNA includes 2'-O-methyl-uridine and 5-methyl-cytosine (m5C). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A). In some embodiments, the mRNA comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In certain embodiments, the mRNAs of the present disclosure are uniformly modified for a particular modification (ie, fully modified, modified throughout the entire sequence). For example, mRNA can be uniformly modified with N1-methylpseudouridine (m1ψ) or 5-methyl-cytidine (m5C), meaning that all uridines or all cytosine nucleosides in the mRNA sequence are modified by N1-methyl Substituted pseudouridine (m1ψ) or 5-methyl-cytidine (m5C). Similarly, for any type of nucleoside residues present in the sequence, the mRNAs of the present disclosure can be consistently modified by substitution with modified residues, such as those listed above.

In some embodiments, an mRNA of the present disclosure may be modified in a coding region (eg, an open reading frame encoding a polypeptide). In other embodiments, the mRNA may be modified in regions other than the coding region. For example, in some embodiments, 5'-UTR and/or 3'-UTR are provided, either or both of which may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the codeable region.

The mmRNA of the present disclosure can include a combination of modifications to sugars, nucleobases, and/or internucleoside linkages. These combinations may include any one or more of the modifications described herein.

Where individual modifications are listed, the nucleosides or nucleotides listed represent 100% of the A, U, G or C nucleotides or nucleosides that have been modified. Where percentages are listed, these represent the percentage of a particular A, U, G or C triphosphate nucleobase in the total amount of A, U, G or C triphosphates present. For example, the combination: 25% 5-aminoallyl-CTP + 75% CTP/25% 5-methoxy-UTP + 75% UTP refers to the following polynucleotides, where 25% of cytosine triphosphates are 5 -Aminoallyl-CTP, while 75% of cytosines are CTP; and 25% of uracils are 5-methoxy UTP, while 75% of uracils are UTP. Where no modified UTP is listed, naturally occurring ATP, UTP, GTP and/or CTP are used at 100% of the sites of those nucleotides present in the polynucleotide. In this example, all GTP and ATP nucleotides were unmodified.

The mRNAs or regions thereof of the present disclosure may be codon-optimized. Codon optimization methods are known in the art and can be used for a variety of purposes: match codon frequencies in the host organism to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize possible damage Tandem repeats of codons or base runs for gene construction or expression, custom transcriptional and translational control regions, insertion or removal of protein trafficking sequences, removal/addition of post-translational modification sites (e.g. glycosylation) in encoded proteins sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, adjust translation rates to allow different domains of the protein to fold correctly, or reduce or Eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms and services are known in the art; non-limiting examples include services of GeneArt (Life Technologies), DNA 2.0 (MenloPark, CA) and/or proprietary methods. In some embodiments, an optimization algorithm is used to optimize the mRNA sequence, eg, to optimize expression in mammalian cells or to enhance mRNA stability.

In certain embodiments, the present disclosure includes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the polynucleotide sequences described herein of polynucleotides.

The mRNAs of the present disclosure can be produced by methods available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid phase, liquid phase, combinatorial synthesis methods, small area synthesis and ligation methods can be used. In some embodiments, the mRNA is prepared using an IVT enzymatic synthesis method. Accordingly, the present disclosure also includes polynucleotides, such as DNA, constructs and vectors, useful for in vitro transcription of the mRNAs described herein.

Non-naturally modified nucleobases can be introduced into polynucleotides, such as mRNA, during or after synthesis. In some embodiments, modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugars. In certain embodiments, modifications can be introduced at the end of the polynucleotide chain or at any other position in the polynucleotide chain; using chemical synthesis or using polymerases.

Enzymatic or chemical ligation methods can be used to conjugate polynucleotides or regions thereof to different functional moieties such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Therapeutic agents for reducing protein expression

In some embodiments, the therapeutic agent is one that reduces (ie, reduces, inhibits, downregulates) protein expression. Non-limiting examples of the types of therapeutics that can be used to reduce protein expression include mRNA, microRNA (miRNA), antagomir, small (short) interfering RNA (siRNA) (including shortmers) that incorporate a microRNA binding site (miR binding site). and dicer-substrate RNA), RNA interference (RNAi) molecules, antisense RNA, ribozymes, small hairpin RNA (shRNA), locked nucleic acid (LNA) and CRISPR/Cas9 technologies.

Peptide/Peptide Therapeutics

In some embodiments, the therapeutic agent is a peptide therapeutic. In some embodiments, the therapeutic agent is a polypeptide therapeutic agent.

In some embodiments, the peptide or polypeptide is naturally derived, eg, isolated from a natural source. In other embodiments, the peptide or polypeptide is a synthetic molecule, eg, a synthetic peptide or polypeptide produced in vitro. In some embodiments, the peptide or polypeptide is a recombinant molecule. In some embodiments, the peptide or polypeptide is a chimeric molecule. In some embodiments, the peptide or polypeptide is a fusion molecule. In some embodiments, the peptide or polypeptide therapeutic agent of the composition is a naturally occurring peptide or polypeptide. In some embodiments, the peptide or polypeptide therapeutic of the composition is a modified version of a naturally occurring peptide or polypeptide (eg, contains less than 3, less than 5, less than 10, less than 15, less than 20 or less than 25 amino acid substitutions, deletions or additions).

In some embodiments, in the loaded LNPs of the present disclosure, the one or more therapeutic and/or prophylactic agents are polynucleotides or polypeptides.

other components

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) may contain one or more components other than those described in the preceding sections. For example, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can comprise one or more small hydrophobic molecules, such as vitamins (eg, vitamin A or vitamin E) or sterols.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) may also contain one or more permeability enhancing molecules, carbohydrates, polymers, surface altering agents, or other components. Carbohydrates can include monosaccharides (eg, glucose) and polysaccharides (eg, glycogen and its derivatives and analogs).

The polymer can be included in and/or used to encapsulate or partially encapsulate the nanoparticle composition. The polymers can be biodegradable and/or biocompatible. The polymer may be selected from, but is not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, Polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile and polyarylates. For example, the polymer may include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) ( PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-propylene) lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D, L-lactide-co-caprolactone-co-glycolide) , L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polycyano Alkyl Acrylate, Polyurethane, Poly-L-Lysine (PLL), Hydroxypropyl Methacrylate (HPMA), Polyethylene Glycol, Poly-L-Glutamic Acid, Poly(hydroxyacid), Polyanhydride, Polyortholic Acid esters, poly(esteramides), polyamides, poly(ester ethers), polycarbonates, polyolefins such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides ( PEO), polyethylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl acetate) (vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), polysiloxane, polystyrene (PS), polyurethane, derivatized cellulose such as alkyl cellulose, hydroxyalkyl cellulose, cellulose ethers, Cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly((meth)methylacrylate) (PMMA), poly(ethyl(meth)acrylate) , poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(((meth)acrylate) lauryl meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and its copolymers and mixtures, polydioxanone and its copolymers, polyhydroxyalkanoates, polytrimethylene fumarate, polyoxymethylene, poloxamer, polyoxyamine, poly(ortho) Esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM), poly(2-methyl) oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), and polyglycerol.

Surface-altering agents may include, but are not limited to, anionic proteins (eg, bovine serum albumin), surfactants (eg, cationic surfactants such as dimethyldi-octadecyl-ammonium bromide), sugars, or sugar derivatives (eg cyclodextrins), nucleic acids, polymers (eg heparin, polyethylene glycol and poloxamers), mucolytics (eg acetylcysteine, mugwort, bromelain, papain, Daqing Clerodendrum, bromhexine, carbocisteine, epraxinone, mesna, ambroxol, sobrerol, Domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta4, dornasealfa ), neltenexine and erdosteine) and DNases (eg rhDNase). Surface-altering agents can be disposed within the nanoparticles and/or on the surface of lipid nanoparticles (eg, empty LNPs or loaded LNPs) (eg, by coating, adsorption, covalent attachment, or other processes).

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can also comprise one or more functionalized lipids. For example, lipids can be functionalized with alkynyl groups that may undergo cycloaddition reactions when exposed to azides under appropriate reaction conditions. In particular, lipid bilayers can be functionalized in this manner with one or more groups that can be used to facilitate membrane penetration, cell recognition or imaging. The surface of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can also be conjugated to one or more useful antibodies. Functional groups and conjugates useful for targeted cellular delivery, imaging and membrane penetration are well known in the art.

In addition to these components, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can contain any substance useful in pharmaceutical compositions. For example, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can comprise one or more pharmaceutically acceptable excipients or adjunct ingredients, such as, but not limited to, one or more solvents, dispersion media, diluents , dispersing aids, suspending aids, granulation aids, disintegrating agents, fillers, glidants, liquid vehicles, binders, surfactants, isotonic agents, thickeners or emulsifiers, buffers , lubricants, oils, preservatives and other substances. Excipients such as waxes, butters, colouring agents, coatings, flavouring and perfuming agents may also be included.

月桂基硫酸钠、季铵化合物和/或其组合。Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, Sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and/or combinations thereof. Granulating and dispersing agents may be selected from a non-limiting list consisting of: potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resin, calcium carbonate, silicate, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl Base cellulose, croscarmellose sodium, methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate

Sodium lauryl sulfate, quaternary ammonium compounds, and/or combinations thereof.

[硅酸镁铝])、长链氨基酸衍生物、高分子量醇类(例如硬脂醇、鲸蜡醇、油醇、单硬脂酸三乙酸甘油酯、二硬脂酸乙二醇酯、单硬脂酸甘油酯、以及单硬脂酸丙二醇酯、聚乙烯醇)、卡波姆(例如羧基聚亚甲基、聚丙烯酸、丙烯酸聚合物、以及羧乙烯聚合物)、卡拉胶、纤维素衍生物(例如羧甲基纤维素钠、粉末状纤维素、羟甲基纤维素、羟丙基纤维素、羟丙基甲基纤维素、甲基纤维素)、脱水山梨糖醇脂肪酸酯(例如，聚氧乙烯脱水山梨糖醇单月桂酸酯

聚氧乙烯脱水山梨糖醇

聚氧乙烯脱水山梨糖醇单油酸酯

脱水山梨糖醇单棕榈酸酯

脱水山梨糖醇单硬脂酸酯

脱水山梨糖醇三硬脂酸酯

单油酸甘油酯、脱水山梨糖醇单油酸酯

聚氧乙烯酯(例如聚氧乙烯单硬脂酸酯

聚氧乙烯氢化蓖麻油、聚乙氧基化蓖麻油、聚甲醛硬脂酸酯以及

)、蔗糖脂肪酸酯、聚乙二醇脂肪酸酯(例如

)、聚氧乙烯醚(例如聚氧乙烯月桂基醚

聚(乙烯吡咯烷酮)、二乙二醇单月桂酸酯、三乙醇胺油酸酯、油酸钠、油酸钾、油酸乙酯、油酸、月桂酸乙酯、月桂基硫酸钠、

溴化十六烷基三甲基铵、氯化十六烷基吡啶、苯扎氯铵、多库酯钠和/或其组合。Surfactants and/or emulsifiers may include, but are not limited to, natural emulsifiers (eg, gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin) , gelatin, egg yolk, casein, lanolin, cholesterol, waxes and lecithin), colloidal clays (such as bentonite [aluminum silicate] and

[Magnesium Aluminum Silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, mono Glyceryl stearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (eg, carboxypolymethylene, polyacrylic acid, acrylic polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives substances (such as sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), sorbitan fatty acid esters (such as , polyoxyethylene sorbitan monolaurate

polyoxyethylene sorbitan

Polyoxyethylene sorbitan monooleate

Sorbitan Monopalmitate

Sorbitan Monostearate

Sorbitan Tristearate

Glyceryl Monooleate, Sorbitan Monooleate

Polyoxyethylene esters (such as polyoxyethylene monostearate

Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and

), sucrose fatty acid esters, polyethylene glycol fatty acid esters (such as

), polyoxyethylene ethers (such as polyoxyethylene lauryl ether

Poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate,

Cetyltrimethylammonium bromide, cetylpyridine chloride, benzalkonium chloride, sodium docusate, and/or combinations thereof.

以及落叶松阿拉伯半乳聚糖)；海藻酸盐；聚环氧乙烷；聚乙二醇；无机钙盐；硅酸；聚甲基丙烯酸酯；蜡；水；醇及其组合，或任何其他适合的粘结剂。Binders can be starches (eg cornstarch and starch paste); gelatin; sugars (eg sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (eg Gum arabic, sodium alginate, carrageenan extract, panwar gum, gum india, mucilage of isapol bark, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl Cellulose, Hydroxypropyl Methylcellulose, Microcrystalline Cellulose, Cellulose Acetate, Poly(vinylpyrrolidone), Magnesium Aluminum Silicate

and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols and combinations thereof, or any other suitable binder.

对羟基苯甲酸甲酯、

NEOLONE^TM、KATHON^TM和/或

Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and/or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate , sodium ascorbate, sodium bisulfite, sodium metabisulfite and/or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, Tartaric acid and/or trisodium edetate. Examples of antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorine Cresol, chloroxylenol, cresol, ethanol, glycerol, hexamidine, imidazolium, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol and/or thimerosal. Examples of antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, sorbitan Potassium acid, sodium benzoate, sodium propionate and/or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and/or phenethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroximemesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Ethylenediamine, Sodium Lauryl Sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulfite, Potassium Metabisulfite, GLYDANT

Methylparaben,

NEOLONE ^™ , KATHON ^™ and/or

Examples of buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronic acid, glucoheptonic acid Calcium, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxyphosphate, acetic acid Potassium, Potassium Chloride, Potassium Gluconate, Potassium Mixture, Dipotassium Hydrogen Phosphate, Potassium Dihydrogen Phosphate, Potassium Phosphate Mixture, Sodium Acetate, Sodium Bicarbonate, Sodium Chloride, Sodium Citrate, Sodium Lactate, Disodium Hydrogen Phosphate, Sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, sulfamate buffer (eg HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's Ringer's solution, ethanol, and/or combinations thereof. Lubricants may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, benzene Sodium formate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

Examples of oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, canola oil, parsley Oil, Carnauba Oil, Castor Oil, Cinnamon Oil, Cocoa Butter Oil, Coconut Oil, Cod Liver Oil, Coffee Oil, Corn Oil, Cottonseed Oil, Emu Oil, Eucalyptus Oil, Evening Primrose Oil, Fish Oil, Flaxseed Oil, Geraniol Oil, Gourd Oil, Grapeseed Oil, Hazelnut Oil, Hyssop Oil, Isopropyl Myristate, Jojoba Oil, Macadamia Nut Oil, Striking Lavender Oil, Lavender Oil, Lemon Oil, Wild Goat Oil, Macadamia Nut Kernel Oil, Mallow Oil, Mango Seed Oil, Mango Seed Oil, Mink Oil, Nutmeg Oil, Olive Oil, Orange Oil, Tangerine Snapper Oil, Palm Oil, Palm Kernel Oil, Peach Kernel Oil, Peanut Oil, Poppy Seed Oil , Pumpkin Seed Oil, Rapeseed Oil, Rice Bran Oil, Rosemary Oil, Safflower Oil, Sandalwood Oil, Camellia Oil, Savory Oil, Sea Buckthorn Oil, Sesame Oil, Shea Butter, Silicone Oil, Soybean Oil, Sunflower Oil, Tea Tree Oils, Thistle Oil, Toona Oil, Vetiver Oil, Walnut Oil and Wheat Germ Oil as well as Butyl Stearate, Caprylic Triglyceride, Capric Triglyceride, Cyclomethicone, Diethyl Sebacate, Dimethicone 360, simethicone, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.

preparation

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can comprise lipid components and one or more additional components, such as therapeutic and/or prophylactic agents. Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be designed for one or more specified applications or targets. Compositions of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be selected based on a particular application or target and/or based on efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more of the components. Similarly, a particular formulation of a nanoparticle composition can be selected for a particular application or target based on, for example, the efficacy and toxicity of a particular combination of ingredients.

The lipid component of the nanoparticle composition may comprise, for example, according to formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y) , (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) Lipids, phospholipids (such as unsaturated lipids, eg, DOPE or DSPC), PEG lipids, and structured lipids. Components of lipid components can be provided in specified fractions.

In some embodiments, the lipid component of the nanoparticle composition comprises according to formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX) , (IY), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) Lipids, phospholipids, PEG lipids and structured lipids. In certain embodiments, the lipid component of the nanoparticle composition comprises from about 30 mol% to about 60 mol% of formulae (A), (A1), (A2), (A3), (B), (I), ( I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), ( II), (IIa) or (IIb), about 0 mol% to about 30 mol% phospholipids, about 18.5 mol% to about 48.5 mol% structured lipids, and about 0 mol% to about 10 mol% PEG lipids, provided that The total mol% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition comprises from about 35 mol% to about 55 mol% of formula (A), (A1), (A2), (A3), (B), (I), ( I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), ( A compound of II), (IIa) or (IIb), about 5 mol% to about 25 mol% phospholipids, about 30 mol% to about 40 mol% structured lipids, and about 0 mol% to about 10 mol% PEG lipids. In a specific embodiment, the lipid component comprises about 50 mol % of the compound, about 10 mol % phospholipids, about 38.5 mol % structured lipids, and about 1.5 mol % PEG lipids. In another specific embodiment, the lipid component comprises about 40 mol % of the compound, about 20 mol % phospholipids, about 38.5 mol % structured lipids, and about 1.5 mol % PEG lipids. In some embodiments, the phospholipid can be DOPE or DSPC. In other embodiments, the PEG lipid can be PEG-1 or PEG _2k -DMG, and/or the structural lipid can be cholesterol.

In some embodiments, empty lipid nanoparticles (empty LNPs) comprise formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb), phospholipids, structured lipids and PEG lipids.

In some embodiments, the loaded lipid nanoparticles (loaded LNPs) comprise formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( The compound of IIb), phospholipids, structured lipids, PEG lipids, and one or more therapeutic and/or prophylactic agents.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I' in an amount of about 40% to about 60% ), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II) , (IIa) or (IIb).

In some embodiments, empty LNPs or loaded LNPs comprise phospholipids in an amount from about 0% to about 20%. For example, in some embodiments, the empty LNP or loaded LNP comprises DSPC in an amount from about 0% to about 20%.

In some embodiments, empty LNPs or loaded LNPs comprise structured lipids in an amount from about 30% to about 50%. For example, in some embodiments, empty LNPs or loaded LNPs comprise cholesterol in an amount from about 30% to about 50%.

In some embodiments, empty LNPs or loaded LNPs comprise PEG lipids in an amount from about 0% to about 5%. For example, in some embodiments, empty LNPs or loaded LNPs comprise PEG-1 or _PEG2k -DMG in an amount from about 0% to about 5%.

In some embodiments, the empty LNP or loaded LNP comprises from about 40 mol % to about 60 mol % of formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), ( A compound of IIa) or (IIb), about 0 mol% to about 20 mol% phospholipids, about 30 mol% to about 50 mol% structured lipids, and about 0 mol% to about 5 mol% PEG lipids.

In some embodiments, the empty LNP or loaded LNP comprises from about 40 mol % to about 60 mol % of formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), ( A compound of IIa) or (IIb), about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG _2k -DMG. In some embodiments, the empty LNP or loaded LNP comprises about 40 mol% to about 60 mol% of the compound of Table 1, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% %PEG _2k -DMG.

In some embodiments, the empty LNP or loaded LNP comprises from about 40 mol % to about 60 mol % of formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), ( A compound of IIa) or (IIb), about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% PEG-1. In some embodiments, the empty LNP or loaded LNP comprises about 40 mol% to about 60 mol% of the compound of Table 1, about 0 mol% to about 20 mol% DSPC, about 30 mol% to about 50 mol% cholesterol, and about 0 mol% to about 5 mol% %PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , phospholipids, structured lipids and PEG lipids, wherein the phospholipid is DSPC and the structured lipid is cholesterol. In some embodiments, the empty LNP or loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the structured lipid is cholesterol.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , a phospholipid, a structured lipid, and a PEG lipid, wherein the structured lipid is cholesterol and the PEG lipid is PEG _2k -DMG. In some embodiments, the empty LNP or loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the structured lipid is cholesterol and the PEG lipid is PEG _2k -DMG.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , phospholipids, structured lipids and PEG lipids, wherein the structured lipid is cholesterol and the PEG lipid is PEG-1. In some embodiments, the empty LNP or loaded LNP comprises the compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the structured lipid is cholesterol and the PEG lipid is PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , a phospholipid, a structured lipid and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG _2k -DMG. In some embodiments, the empty LNP or loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG _2k -DMG.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , phospholipids, structural lipids and PEG lipids, wherein the phospholipid is DSPC and the PEG lipid is PEG-1. In some embodiments, the empty LNP or loaded LNP comprises the compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the PEG lipid is PEG-1.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , a phospholipid, a structured lipid and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid is PEG _2k -DMG. In some embodiments, the empty LNP or loaded LNP loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid The mass is PEG _2k -DMG.

In some embodiments, an empty LNP or a loaded LNP comprises a compound of formula (A), a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid The mass is PEG _2k -DMG. In some embodiments, the empty LNP or loaded LNP loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid The mass is PEG _2k -DMG.

In some embodiments, the empty LNP or loaded LNP comprises formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) Compounds of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , a phospholipid, a structured lipid and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid is PEG-1.

In some embodiments, an empty LNP or a loaded LNP comprises a compound of formula (A), a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid The quality is PEG-1. In some embodiments, the empty LNP or loaded LNP comprises a compound of Table 1, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, the structured lipid is cholesterol and the PEG lipid is PEG-1.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed to deliver therapeutic and/or prophylactic agents such as RNA to specific cells, tissues, organs or systems or groups thereof in a mammal. To increase selectivity for specific body targets, the physicochemical properties of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be altered. For example, granularity can be adjusted based on the fenestration size of different organs. The therapeutic and/or prophylactic agents included in the nanoparticle composition can also be selected based on one or more desired delivery targets. For example, therapeutic and/or prophylactic agents can be selected for a particular indication, condition, disease or disorder and/or for delivery (eg, local or specific delivery) to a particular cell, tissue, organ or system or group thereof. In certain embodiments, a nanoparticle composition may comprise mRNA encoding a polypeptide of interest that is capable of being translated within a cell to produce the polypeptide of interest. Such compositions can be designed for specific delivery to specific organs. In some embodiments, the composition can be designed for specific delivery to mammalian liver.

The amount of therapeutic and/or prophylactic agent in a nanoparticle composition may depend on the size, composition, desired target and/or application or other characteristics of the nanoparticle composition, as well as the characteristics of the therapeutic and/or prophylactic agent. For example, the amount of RNA that can be used in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and/or prophylactic agents and other ingredients (eg, lipids) in the nanoparticle composition can also vary. In some embodiments, the weight/weight ratio of lipid component to therapeutic and/or prophylactic agent in the nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7 :1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 , 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the weight/weight ratio of lipid component to therapeutic and/or prophylactic agent may be from about 10:1 to about 40:1. In certain embodiments, the weight/weight ratio is about 20:1.

The amount of therapeutic and/or prophylactic agent in a nanoparticle composition can be measured, for example, using absorption spectroscopy (eg, UV-Vis spectroscopy).

In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a particular N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In general, lower N:P ratios are preferred. The one or more RNAs, lipids, and amounts thereof can be selected to provide from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 , 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30 :1 N:P ratio. In certain embodiments, the N:P ratio can be from about 2:1 to about 8:1. In other embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio can be about 5.67:1.

physical properties

The characteristics of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can depend on their components. For example, lipid nanoparticles comprising cholesterol as a structural lipid (eg, empty LNP or loaded LNP) may have different characteristics than lipid nanoparticles comprising different structured lipids (eg, empty LNP or loaded LNP). Similarly, the characteristics of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can depend on the absolute or relative amounts of their components. For example, lipid nanoparticles comprising higher mole fractions of phospholipids (eg, empty LNPs or loaded LNPs) may have different characteristics than lipid nanoparticles comprising lower mole fractions of phospholipids (eg, empty LNPs or loaded LNPs) . Properties can also vary depending on the method and conditions of preparation of the nanoparticle composition.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be characterized by a variety of methods. For example, microscopy (eg, transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and particle size distribution of nanoparticle compositions. Dynamic light scattering or potentiometric analysis (eg potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Various characteristics of nanoparticle compositions, such as particle size, polydispersity index and zeta potential, can also be measured using an instrument such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).

The average size of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be between tens of nanometers and hundreds of nanometers, eg, as measured by dynamic light scattering (DLS). For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135nm, 140nm, 145nm or 150nm. In some embodiments, the lipid nanoparticles (eg, empty LNPs or loaded LNPs) can have an average size of about 50 nm to about 100 nm, about 50 nm to about 90 nm, about 50 nm to about 80 nm, about 50 nm to about 70 nm, about 50 nm to about 60 nm, about 60 nm to about 100 nm, about 60 nm to about 90 nm, about 60 nm to about 80 nm, about 60 nm to about 70 nm, about 70 nm to about 150 nm, about 70 nm to about 130 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, about 70 nm to about 80 nm, about 80 nm to about 150 nm, about 80 nm to about 130 nm, about 80 nm to about 100 nm, about 80 nm to about 90 nm, about 90 nm to about 150 nm, about 90 nm to about 130 nm, or about 90 nm to about 100 nm. In certain embodiments, the lipid nanoparticles (eg, empty LNPs or loaded LNPs) can have an average size of about 70 nm to about 130 nm or about 70 nm to about 100 nm. In a particular embodiment, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm. In other embodiments, the average size can be about 120 nm.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be relatively homogeneous. The polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, such as the particle size distribution of lipid nanoparticles (eg, empty LNPs or loaded LNPs). A smaller (eg, less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12 , 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be from about 0.10 to about 0.20.

The zeta potential of lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Lipid nanoparticles (e.g., empty LNPs or loaded LNPs) with relatively low charges (positive or negative) are generally desirable because higher-charged species may interact with cells, tissues, and other components in the body. hopeful interaction. In some embodiments, the zeta potential of the lipid nanoparticle (eg, empty LNP or loaded LNP) can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +10 mV 10mV to about +5mV, about -10mV to about 0mV, about -10mV to about -5mV, about -5mV to about +20mV, about -5mV to about +15mV, about -5mV to about +10mV, about -5mV to about +5mV, about -5mV to about 0mV, about 0mV to about +20mV, about 0mV to about +15mV, about 0mV to about +10mV, about 0mV to about +5mV, about +5mV to about +20mV, about +5mV to about About +15mV or about +5mV to about +10mV.

The encapsulation efficiency of a therapeutic and/or prophylactic agent describes, relative to the initial amount provided, encapsulated in a nanoparticle composition (eg, empty LNP or loaded LNP) after preparation or otherwise combined with lipids. The amount of therapeutic and/or prophylactic agent associated with the mass nanoparticles. A higher encapsulation efficiency (eg, close to 100%) is desired. Encapsulation efficiency can be measured, for example, by comparing lipid nanoparticles (eg, empty LNPs or loaded LNPs) before and after disruption of lipid nanoparticles (eg, empty LNPs or loaded LNPs) with one or more organic solvents or detergents The amount of therapeutic and/or prophylactic agent in the solution is measured. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic (eg, RNA) in solution. For lipid nanoparticles (eg, empty LNPs or loaded LNPs) described herein, the encapsulation efficiency of therapeutic and/or prophylactic agents can be at least 50%, eg, 50%, 55%, 60%, 65%, 70% %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%. In some embodiments, the encapsulation efficiency of the therapeutic and/or prophylactic agent is between 80% and 100%.

pharmaceutical composition

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be formulated in whole or in part into pharmaceutical compositions. The pharmaceutical composition can include one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs). In one embodiment, the pharmaceutical composition comprises a population of lipid nanoparticles (eg, empty LNPs or loaded LNPs). For example, a pharmaceutical composition can include one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs) that include one or more different therapeutic and/or prophylactic agents. The pharmaceutical compositions may also contain one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidance on the formulation and manufacture of pharmaceutical compositions and agents is available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006. Conventional excipients and adjunct ingredients can be used in any pharmaceutical composition, except that any conventional excipients or adjunct ingredients may be incompatible with one or more components of the nanoparticle composition. Excipients or adjuncts may be associated with the components of the lipid nanoparticle (eg empty LNP or LNP-loaded) if their combination may cause any undesired biological or other detrimental effects. Components are incompatible.

In some embodiments, one or more excipients or adjunct ingredients may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, one or more excipients or adjunct ingredients may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical conventional. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipients are approved for human and veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia.

The one or more lipid nanoparticles (eg, empty LNP or loaded LNP), the one or more pharmaceutically acceptable excipients and/or any additional ingredients in the pharmaceutical composition according to the present disclosure The relative amounts of s will vary according to the identity, size and/or condition of the subject being treated and also according to the route of administration of the composition. For example, a pharmaceutical composition may comprise between 0.1% and 100% (weight/weight) of one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs).

In certain embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) and/or pharmaceutical compositions of the present disclosure are refrigerated or frozen for storage and/or transport (eg, stored at 4°C or lower) , such as a temperature between about -150°C and about 0°C or between about -80°C and about -20°C (eg, about -5°C, -10°C, -15°C, -20°C, - 25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)). For example, including formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), A pharmaceutical composition of a compound of any one of (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) is Solutions, which are stored and/or shipped frozen, eg, at about -20°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C. In certain embodiments, the present disclosure also relates to a method that adds formulas (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2) ), (A-3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), Lipid nanoparticles (eg, empty A method of stability of LNP or loaded LNP) and/or pharmaceutical composition by storing lipid nanoparticles (eg, empty LNP or loaded LNP) and/or pharmaceutical composition at 4°C or lower , such as between about -150°C and about 0°C or between about -80°C and about -20°C, eg, about -5°C, -10°C, -15°C, -20°C, -25°C , -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C). For example, lipid nanoparticles (eg, empty LNPs or loaded LNPs) and/or pharmaceutical compositions disclosed herein are stable, eg, at temperatures of 4°C or less (eg, between about 4°C and -20°C) For about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months , at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months. In some embodiments, the formulation is stable at about 4°C for at least 4 weeks. In certain embodiments, a pharmaceutical composition of the present disclosure comprises a lipid nanoparticle disclosed herein (eg, empty LNP or loaded LNP) and a pharmaceutically acceptable carrier selected from Tris, acetate (eg, sodium acetate), one or more of citrate (eg, sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical compositions of the present disclosure is between about 7 and 8 (eg, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or between 7.5 and 8 or between 7 and 7.8). For example, the pharmaceutical compositions of the present disclosure comprise lipid nanoparticles disclosed herein (eg, empty LNPs or loaded LNPs), Tris, normal saline, and sucrose, and have a pH of about 7.5-8, which is suitable for use at, eg, about -20°C storage and/or transport. For example, a pharmaceutical composition of the present disclosure comprises a lipid nanoparticle disclosed herein (eg, empty LNP or loaded LNP) and PBS, and has a pH of about 7-7.8, which is suitable for storage at, eg, about 4°C or lower. and/or transportation. In the context of the present disclosure, "stable," "stabilized," and "stable" refer to the lipid nanoparticles (eg, empty LNPs or loaded LNPs) and/or pharmaceutical compositions disclosed herein in a given manufacture resistance to chemical or physical changes (eg, degradation, particle size changes, aggregation, encapsulation changes, etc.) sex.

In some embodiments, the pharmaceutical compositions of the present disclosure comprise empty LNP or loaded LNP, a cryoprotectant, a buffer, or a combination thereof.

In some embodiments, the cryoprotectant includes one or more cryoprotectants, and each of the one or more cryoprotectants is independently a polyol (eg, a diol or triol, such as propylene glycol ( i.e., 1,2-propanediol), 1,3-propanediol, glycerol, (+/-)-2-methyl-2,4-pentanediol, 1,6-hexanediol, 1,2-butanediol alcohol, 2,3-butanediol, ethylene glycol, or diethylene glycol), non-detergent sulfobetaines (eg, NDSB-201 (3-(1-pyrido)-1-propanesulfonate) , penetrants (eg, L-proline or trimethylamine N-oxide dihydrate), polymers (eg, polyethylene glycol 200 (PEG 200), PEG 400, PEG 600, PEG 1000, PEG _2k- DMG, PEG 3350, PEG 4000, PEG 8000, PEG 10000, PEG20000, polyethylene glycol monomethyl ether 550 (mPEG 550), mPEG 600, mPEG 2000, mPEG 3350, mPEG 4000, mPEG5000, polyvinylpyrrolidone (e.g. polyvinylpyrrolidone) Pyrrolidone K15), pentaerythritol propoxylate or polypropylene glycol P400), organic solvents (eg, dimethyl sulfoxide (DMSO) or ethanol), sugars (eg, D-(+)-sucrose, D-sorbitol, seaweed) sugar, D-(+)-maltose monohydrate, endoerythritol, xylitol, inositol, D-(+)-raffinose pentahydrate, D-(+)-trehalose dihydrate or D-(+)-glucose monohydrate), or salts (e.g., lithium acetate, lithium chloride, lithium formate, lithium nitrate, lithium sulfate, magnesium acetate, sodium acetate, sodium chloride, sodium formate, sodium malonate, sodium nitrate, sodium sulfate, or any hydrate thereof) or any combination thereof. In some embodiments, the cryoprotectant includes sucrose. And in some embodiments, the cryoprotectant and/or excipient is sucrose. In some implementations In embodiments, the cryoprotectant includes sodium acetate. And in some embodiments, the cryoprotectant and/or excipient is sodium acetate. In some embodiments, the cryoprotectant includes sucrose and sodium acetate.

In some embodiments, wherein the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, tris buffer, and combinations thereof.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) and/or pharmaceutical compositions comprising one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be administered to any patient or subject, Included are patients or subjects who may benefit from the therapeutic effect provided by the delivery of therapeutic and/or prophylactic agents to one or more specific cells, tissues, organs or systems or groups thereof. Although the descriptions provided herein with respect to lipid nanoparticles (eg, empty LNPs or loaded LNPs) and pharmaceutical compositions comprising lipid nanoparticles (eg, empty LNPs or loaded LNPs) are primarily directed to compositions suitable for administration to humans, However, the skilled artisan will appreciate that such compositions are generally suitable for administration to any other mammal. It is fully understood that compositions suitable for administration to humans can be modified to make the compositions suitable for administration to a variety of animals, and can be devised and devised by the ordinarily skilled veterinary pharmacologist with only routine experimentation, if any. /or make such modifications. Subjects to which the compositions are expected to be administered include, but are not limited to, humans, other primates and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and/or or rats. The lipid nanoparticles of the present invention can also be used in in vitro and ex vivo applications.

Pharmaceutical compositions comprising one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be prepared by any method known in the art of pharmacology or later developed. In general, such methods of preparation involve bringing into association the active ingredient with excipients and/or one or more other accessory ingredients, and then dividing, shaping and/or packaging the product as desired or necessary into the desired Single-dose unit or multiple-dose unit.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk in a single unit dose and/or in a plurality of single unit doses. As used herein, a "unit dose" is a discrete quantity of a pharmaceutical composition containing a predetermined quantity of an active ingredient (eg, a nanoparticle composition). The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject and/or a suitable fraction of such dose, such as, for example, one-half or one-third of such dose.

Pharmaceutical compositions can be prepared in a variety of forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (eg, emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (eg, capsules, tablets, pills, powders and granules), dosage forms for topical and/or transdermal administration (eg, ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), mixed Suspensions, powders and other forms.

醇、油、改性油、二醇、聚山梨醇酯、环糊精、聚合物和/或其组合混合。Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents commonly used in the art, such as, for example, water or other solvents; solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol , benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydro Fatty acid esters of furfuryl alcohol, polyethylene glycol and sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also contain additional therapeutic and/or prophylactic agents, additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and/or perfuming agents. In certain embodiments for parenteral administration, the composition is combined with a solubilizer such as

Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof are mixed.

Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension and/or emulsion in a non-toxic parenterally acceptable diluent and/or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution (U.S.P.) and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid are useful in the preparation of injectables.

Injectable preparations can be prepared, for example, by filtration through a bacterial-retaining filter and/or by introducing a sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Sterilize.

In order to prolong the effect of the active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by using liquid suspensions of poorly water-soluble crystalline or amorphous materials. The rate of absorption of a drug depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are prepared by forming microencapsulation matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are usually suppositories, which may be prepared by mixing the composition with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol, or a suppository wax. Excipients are solid at ambient temperature but liquid at body temperature and will therefore melt in the rectal or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, films, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dibasic calcium phosphate and/or fillers or bulking agents (eg, starch, lactose, sucrose, glucose, mannitol, and silicic acid), binders (eg, carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), humectants (eg, glycerol), disintegrants ( For example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate), dissolution delaying agents (eg, paraffin), absorption enhancers (eg, quaternary ammonium compounds), wetting agents ( For example, cetyl alcohol and glycerol monostearate), adsorbents (for example, kaolin and bentonite, silicates) and lubricants (for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl sodium sulfate) and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may contain buffering agents.

Similar types of solid compositions can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and may also be of a composition that they release the active ingredient only or preferentially in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of the compositions may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and/or any desired preservatives and/or buffers that may be required. Additionally, the present disclosure contemplates the use of transdermal patches, which generally have the added advantage of providing controlled delivery of compounds to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispersing the compound in the proper medium. Alternatively or additionally, the rate can be controlled by providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

Suitable devices for delivering the intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by means of devices that limit the effective depth of penetration of the needle into the skin. Jet injection devices are suitable which deliver the liquid composition to the dermis via a liquid jet injector and/or via a needle that penetrates the stratum corneum and produces a jet that reaches the dermis. Ballistic powder/particle delivery devices are suitable which use compressed gas to accelerate the vaccine in powder form across the outer layers of the skin to the dermis. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration.

Formulations suitable for topical application include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspension. For example, a topically administrable formulation may contain from about 1% to about 10% (weight/weight) active ingredient, although the active ingredient may be present in concentrations up to the solubility limit of the active ingredient in the solvent. Formulations for topical administration may also contain one or more of the additional ingredients described herein.

Pharmaceutical compositions can be prepared, packaged and/or sold in formulations suitable for pulmonary administration via the oral cavity. Such formulations may contain dry particles containing the active ingredient. Such compositions are conveniently in dry powder form for application using a device comprising a dry powder reservoir from which the propellant flow can be directed to dispense powder; and/or using a self-propelling solvent/powder dispensing container, such as in a sealed container. The container contains a device for dissolving and/or suspending the active ingredient in a low boiling point propellant. Dry powder compositions may include a solid fine powder diluent such as sugar, and are conveniently presented in unit dosage form.

Low boiling point propellants generally include liquid propellants that boil below 65°F at large ambient pressures. In general, the propellant may comprise from 50% to 99.9% (w/w) of the composition and the active ingredient may comprise from 0.1% to 20% (w/w) of the composition. The propellant may also contain additional ingredients such as liquid nonionic and/or solid anionic surfactants and/or solid diluents (which may have a particle size of the same order of magnitude as the particles containing the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged and/or sold as optionally sterile, aqueous and/or dilute alcoholic solutions and/or suspensions containing the active ingredient, and any spraying and/or atomizing device may be used. Easy to apply. Such formulations may also contain one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants and/or preservatives such as methyl hydroxybenzoate. The average diameter of the droplets provided by this route of administration can range from about 1 nm to about 200 nm.

Formulations described herein as useful for pulmonary delivery can be used for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. This formulation is administered by snuff, ie by rapid inhalation through the nasal passages from a powder container near the nose.

Formulations suitable for nasal administration may, for example, contain as little as about 0.1% (w/w) and as high as 100% (w/w) active ingredient, and may contain one or more of the additional ingredients described herein. Pharmaceutical compositions can be prepared, packaged and/or sold in formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges prepared using conventional methods, and may contain, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising orally dissolvable and/or degradable The composition and optional one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may include powders and/or aerosolized and/or aerosolized solutions and/or suspensions containing the active ingredient. Such powdered, aerosolized and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range of about 0.1 nm to about 200 nm, and may also contain any of the additional ingredients described herein. one or more of.

Pharmaceutical compositions can be prepared, packaged and/or sold in formulations suitable for ocular administration. Such formulations may, for example, be in the form of eye drops containing, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such drops may also contain one or more of buffers, salts, and/or any of the additional ingredients described herein. Other ophthalmically administrable formulations that can be used include those comprising the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are intended to be within the scope of this disclosure.

mRNA therapy

mRNA in drug form has the potential to deliver secreted proteins as well as intracellular and transmembrane proteins. mRNA in drug form has the potential to deliver transmembrane and intracellular proteins, targets that are inaccessible to standard biologics when delivered in protein form due to their inability to cross the cell membrane. A major challenge in making mRNA-based therapies a reality is the identification of optimal delivery vehicles. Due to the large size, chemical instability and potential immunogenicity of mRNA, mRNA requires a delivery vehicle that can provide protection from endo- and exonuclease and protect the cargo from attack by immune sentinels thing. Lipid nanoparticles (LNPs) have been identified as the primary choice in this regard.

A key performance criterion for lipid nanoparticle delivery systems is to maximize cellular uptake and enable efficient mRNA release from endosomes. In one embodiment, the LNPs of the invention comprising the novel lipids disclosed herein exhibit improvements in at least one of cellular uptake and endosomal release. At the same time, the LNP must provide a stable drug product that can be safely administered at therapeutically relevant levels. LNPs are multi-component systems that typically consist of amino lipids, phospholipids, cholesterol, and PEG-lipids. Each component is necessary for efficient delivery and particle stability of nucleic acid cargo. The key components thought to drive cellular uptake, endosomal escape and tolerance are amino lipids. Cholesterol and PEG-lipids contribute to in vivo stability and shelf stability of the drug product, while phospholipids provide additional fusion with LNPs and thus help drive endosomal escape and make nucleic acids bioavailable in the cytosol.

Over the past few decades, several aminolipid families have been developed for oligonucleotide delivery, including the aminolipid MC3 (DLin-MC3-DMA). MC3-based LNPs have been shown to deliver mRNA efficiently. Such LNPs are rapidly opsonized by apolipoprotein E (ApoE) upon intravenous delivery, enabling their uptake by low-density lipoprotein receptor (LDLr) cells. However, concerns remain that the long tissue half-life of MC3 may lead to undesirable side effects, preventing its use in chronic therapy. Furthermore, extensive literature evidence suggests that long-term administration of lipid nanoparticles produces several toxic side effects, including complement activation-associated anaphylactoid reactions (CARPA) and liver damage. Therefore, to unlock the potential of mRNA and other nucleic acid, nucleotide or peptide-based therapeutics in humans, a class of LNPs with improved delivery efficiency as well as metabolic and toxicity profiles that enable long-term administration in humans is required.

The ability to treat a wide range of diseases requires the flexibility of long-term safe dosing at different dose levels. Through systematic optimization of aminolipid structures, the disclosed compounds were identified as compounds that balance chemical stability, enhanced delivery efficiency due to improved endosomal escape, rapid in vivo metabolism, and a clean toxicity profile. The combination of these features will provide drug candidates that can be administered chronically without activating the immune system. Initial rodent screening allowed the identification of lead lipids with good delivery efficiency and pharmacokinetics. The delivery efficiency of lead LNPs after single and repeated dosing was further analyzed in non-human primates. Finally, the optimized LNPs were evaluated in rats and non-human primates in a one-month repeated-dose toxicity study. Without wishing to be bound by theory, the novel ionizable lipids of the present disclosure have improved cellular delivery, improved protein expression, and improved biodegradability properties, which may result in higher levels of ionization in cells compared to LNPs lacking the lipids of the present invention. mRNA expression was increased by more than 2-fold, 5-fold, 10-fold, 15-fold or 20-fold. In another embodiment, LNPs comprising the lipids of the present invention can result in specific (eg, preferential) delivery to one or more cell types compared to other cell types, resulting in a In some cells or tissues, mRNA expression is increased by more than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold compared to high-quality LNPs. These improvements over existing technologies allow for the safe and effective use of mRNA-based therapies in acute and chronic diseases.

method

In some aspects, the present disclosure provides a method of delivering therapeutic and/or prophylactic agents to cells (eg, mammalian cells). This method includes the step of contacting a cell with the LNP-loaded or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and/or prophylactic agent to the cell. In some embodiments, the cells are in the subject and the contacting comprises administering the cells to the subject. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb), phospholipids, structured lipids, PEG lipids and lipid nanoparticles of one or more therapeutic and/or prophylactic agents, thereby delivering said therapeutic and/or prophylactic agents to said Cell steps.

In some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1) , (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic) , (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG _2k -DMG and one or more selected from nucleotides Steps of lipid nanoparticles of therapeutic and/or prophylactic agents, polypeptides, and nucleic acids (eg, RNA). For example, in some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a compound comprising formula (A) , DSPC, cholesterol and PEG _2k -DMG and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1) , (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic) , (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG-1 and one or more selected from the group consisting of nucleotides, Lipid Nanoparticles of Polypeptides and Nucleic Acids (eg, RNA) for Therapeutic and/or Prophylactic Agents. For example, in some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to cells in a subject, wherein the method comprises administering to the subject a compound comprising formula (A) , DSPC, cholesterol and PEG-1 and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some aspects, the present disclosure provides a method of delivering (eg, specific delivery) a therapeutic and/or prophylactic agent to a mammalian organ or tissue (eg, liver, kidney, spleen, or lung). This method includes the step of contacting the cells with the LNP-loaded or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and/or prophylactic agent to the target organ or tissue. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb), phospholipids, structured lipids, PEG lipids and lipid nanoparticles of one or more therapeutic and/or prophylactic agents, thereby delivering said therapeutic and/or prophylactic agents to said A step in a target organ or tissue.

In some embodiments, the present disclosure provides a method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1) ), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic ), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG _2k -DMG and one or more selected from the group consisting of nucleosides Steps of lipid nanoparticles of therapeutic and/or prophylactic agents of acids, polypeptides and nucleic acids (eg, RNA). For example, in some embodiments, the present disclosure provides a method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject a compound comprising formula (A) The step of compound, DSPC, cholesterol and PEG _2k -DMG and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), Compounds of (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb), DSPC, cholesterol and PEG-1 and one or more selected from nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic lipid nanoparticles steps. For example, in some embodiments, the present disclosure provides a method of delivering a therapeutic and/or prophylactic agent to an organ of a subject, wherein the method comprises administering to the subject a compound comprising formula (A), The step of DSPC, cholesterol and PEG-1 and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some aspects, the disclosure features a method for enhanced delivery of a therapeutic and/or prophylactic agent (eg, mRNA) to a target tissue (eg, liver, spleen, or lung). Such methods include contacting the cells with an LNP-loaded or pharmaceutical composition of the present disclosure, thereby delivering the therapeutic and/or prophylactic agents to the target tissue (eg, liver, kidney, spleen, or lung) step. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb), phospholipids, structured lipids, PEG lipids and lipid nanoparticles of one or more therapeutic and/or prophylactic agents, thereby delivering said therapeutic and/or prophylactic agents to said Steps to target tissues (eg, liver, kidney, spleen, or lung).

In some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue, wherein the method comprises administering to a subject a method comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), A compound of (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb), DSPC, cholesterol and PEG _2k -DMG and one or more selected from the group consisting of nucleotides, Lipid Nanoparticles of Polypeptides and Nucleic Acids (eg, RNA) for Therapeutic and/or Prophylactic Agents. For example, in some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue, wherein the method comprises administering to a subject a compound comprising formula (A), A step of DSPC, cholesterol and PEG _2k -DMG and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue, wherein the method comprises administering to a subject a method comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), Compounds of (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb), DSPC, cholesterol and PEG-1 and one or more selected from nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic lipid nanoparticles steps. For example, in some embodiments, the present disclosure provides a method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue, wherein the method comprises administering to a subject a compound comprising formula (A), The step of DSPC, cholesterol and PEG-1 and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents.

In some aspects, the present disclosure provides a method of producing a polypeptide of interest in a cell (eg, a mammalian cell). This method includes the step of contacting a cell with a loaded LNP or pharmaceutical composition of the present disclosure, wherein the loaded LNP or pharmaceutical composition comprises mRNA, whereby the mRNA is capable of being translated in the cell to produce a polypeptide. In some embodiments, the cells are in the subject and the contacting comprises administering the cells to the subject. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( The compound of IIb), the phospholipid, the structured lipid, the PEG lipid and the step of lipid nanoparticles of mRNA, whereby the mRNA can be translated in the cell to produce the polypeptide.

In some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a method comprising formula (A), (A1), (A2), (A3) , (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) Steps for lipid nanoparticles of compounds, (If), (Ig), (II), (IIa) or (IIb), DSPC, cholesterol and PEG _2k -DMG and mRNA. For example, in some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a compound comprising Table 1, DSPC, cholesterol, and PEG _2k -DMG, and Steps for mRNA lipid nanoparticles. For example, in some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a compound comprising formula (A), DSPC, cholesterol, and PEG _2k- Steps for DMG and mRNA lipid nanoparticles.

In some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a method comprising formula (A), (A1), (A2), (A3) , (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) Steps for lipid nanoparticles of compounds, (If), (Ig), (II), (IIa) or (IIb), DSPC, cholesterol and PEG-1 and mRNA. For example, in some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a compound comprising formula (A), DSPC, cholesterol, and PEG-1 and the steps of mRNA lipid nanoparticles. For example, in some embodiments, the present disclosure provides a method of producing a polypeptide of interest in a cell, wherein the method comprises administering to a subject a compound comprising Table 1, DSPC, cholesterol and PEG-1, and mRNA steps of lipid nanoparticles.

In some aspects, the present disclosure provides a method of treating a disease or disorder in a mammal (eg, a human) in need thereof. The method includes the step of administering to the mammal a therapeutically effective amount of a loaded LNP or pharmaceutical composition of the present disclosure. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Compounds of IIb), phospholipids, structured lipids, PEG lipids and lipid nanoparticles of one or more therapeutic and/or prophylactic agents, thereby delivering said therapeutic and/or prophylactic agents to said Cell steps. In some embodiments, the disease or disorder is characterized by dysfunctional or aberrant protein or polypeptide activity. For example, the disease or disorder is selected from the group consisting of rare diseases, infectious diseases, cancer and proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and Metabolic disease.

In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1 ), (A2), (A3) , (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) , (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG _2k -DMG and one or more selected from the group consisting of nucleotides, polypeptides and nucleic acids (e.g., RNA ) of lipid nanoparticles of therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a compound comprising formula (A), DSPC, cholesterol, and PEG _2k- The step of DMG and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides, and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a compound comprising Table 1, DSPC, cholesterol, and PEG _2k -DMG, and The step of one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from the group consisting of nucleotides, polypeptides, and nucleic acids (eg, RNA).

In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1 ), (A2), (A3) , (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) , (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG-1 and one or more selected from nucleotides, polypeptides and nucleic acids (eg, RNA) Steps of lipid nanoparticles of therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a compound comprising formula (A), DSPC, cholesterol, and PEG-1 and one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from the group consisting of nucleotides, polypeptides, and nucleic acids (eg, RNA). For example, in some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject, wherein the method comprises administering to the subject a compound comprising Table 1, DSPC, cholesterol and PEG-1 and a The step of one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (eg, RNA).

In yet another aspect, the present disclosure features a method of reducing immunogenicity comprising introducing a loaded LNP or pharmaceutical composition of the present disclosure into a cell, wherein induction of a cellular immune response in the cell induced by the reference composition In contrast, the LNP-loaded or pharmaceutical composition reduces the induction of a cellular immune response by the cells to the LNP-loaded or pharmaceutical composition. In some embodiments, the cells are in the subject and the contacting comprises administering the cells to the subject. In some embodiments, the method comprises administering to the subject a compound comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya)(IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or ( Steps of compounds of IIb), phospholipids, structured lipids, PEG lipids, and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents , wherein compared with the induction of a cellular immune response in cells induced by the reference compound, comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), ( I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa ) or (IIb) compounds of the lipid nanoparticles reduce said cell pair comprising formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X ), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) Induction of cellular immune responses by lipid nanoparticles of compounds or (IIb). For example, the cellular immune response is an innate immune response, an adaptive immune response, or both.

In some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1), (A2), (A3) , (B), (I), (I'), (IX), (IY), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) , (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG _2k -DMG and one or more selected from the group consisting of nucleotides, polypeptides and nucleic acids (e.g., RNA ) of lipid nanoparticles of therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a compound comprising formula (A), DSPC, cholesterol, and PEG _2k- The step of DMG and one or more lipid nanoparticles selected from the group consisting of nucleotides, polypeptides, and nucleic acid (eg, RNA) therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a compound comprising Table 1, DSPC, cholesterol, and PEG _2k -DMG, and The step of one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from the group consisting of nucleotides, polypeptides, and nucleic acids (eg, RNA).

In some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a method comprising formula (A), (A1), (A2), (A3) , (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie) , (If), (Ig), (II), (IIa) or (IIb) compounds, DSPC, cholesterol and PEG-1 and one or more selected from nucleotides, polypeptides and nucleic acids (eg, RNA) Steps of lipid nanoparticles of therapeutic and/or prophylactic agents. For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a compound comprising formula (A), DSPC, cholesterol, and PEG-1 and one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from the group consisting of nucleotides, polypeptides, and nucleic acids (eg, RNA). For example, in some embodiments, the present disclosure provides a method of reducing immunogenicity in a subject, wherein the method comprises administering to the subject a compound comprising Table 1, DSPC, cholesterol and PEG-1 and a The step of one or more lipid nanoparticles of therapeutic and/or prophylactic agents selected from nucleotides, polypeptides, and nucleic acids (eg, RNA).

The present disclosure also includes the synthesis of formulae (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA ), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) compounds, and methods for preparing compounds comprising lipids Methods of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) of lipid components comprising formulae (A), (A1), (A2), (A3), (B), (I) ), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig ), (II), (IIa) or (IIb).

Methods of producing polypeptides in cells

The present disclosure provides methods for producing polypeptides of interest in mammalian cells. Methods of producing polypeptides include contacting cells with lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising mRNA encoding the polypeptide of interest. After the cell is contacted with the nanoparticle composition, the mRNA can be taken up and translated in the cell to produce the polypeptide of interest.

In general, the step of contacting mammalian cells with lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising mRNA encoding a polypeptide of interest can be performed in vivo, ex vivo, in culture, or in vitro. The amount of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) and/or the amount of mRNA therein contacted with the cells can depend on the type of cells or tissues contacted, the mode of administration, the lipid nanoparticles (e.g., empty LNPs). LNPs or loaded LNPs) and the physicochemical characteristics of the mRNAs therein (eg, size, charge, and chemical composition), among other factors. In general, an effective amount of lipid nanoparticles (eg, empty LNPs or loaded LNPs) will allow efficient production of polypeptides in cells. Measures of efficiency can include polypeptide translation (indicated by polypeptide expression), levels of mRNA degradation, and indicators of immune response.

The step of contacting the mRNA-containing lipid nanoparticles (eg, empty LNPs or loaded LNPs) with the cells may include or result in transfection. Phospholipids included in lipid components of lipid nanoparticles (e.g., empty LNPs or loaded LNPs) can facilitate transfection and/or increase transfection efficiency, e.g., by interacting and/or fusing with cell membranes or intracellular membranes . Transfection can allow translation of mRNA within the cell.

In some embodiments, the lipid nanoparticles described herein (eg, empty LNPs or loaded LNPs) can be used therapeutically. For example, mRNA contained in a lipid nanoparticle (eg, empty LNP or loaded LNP) can encode a therapeutic polypeptide (eg, in a translatable region), and upon contacting and/or entering (eg, transfecting) a cell Production of therapeutic polypeptides. In other embodiments, the mRNA contained in the lipid nanoparticle (eg, empty LNP or loaded LNP) can encode a polypeptide that improves or increases immunity in a subject. For example, the mRNA can encode granulocyte colony stimulating factor or trastuzumab.

In certain embodiments, the mRNA contained in the lipid nanoparticle (eg, empty LNP or loaded LNP) can encode a recombinant polypeptide that can replace cells that may not be substantially present in contact with the nanoparticle composition one or more of the polypeptides. The one or more substantially absent polypeptides may be lacking due to mutations in the genes encoding the genes or their regulatory pathways. Alternatively, recombinant polypeptides produced by translation of mRNA can antagonize the activity of endogenous proteins present in the cell, on the cell surface, or secreted by the cell. It may be desirable for antagonistic recombinant polypeptides to counteract deleterious effects caused by the activity of the endogenous protein, such as altered activity or localization caused by mutation. In another alternative, recombinant polypeptides produced by translation of mRNA may indirectly or directly antagonize the activity of biological moieties present in, on the surface of, or secreted by cells. Antagonized biological moieties can include, but are not limited to, lipids (eg, cholesterol), lipoproteins (eg, low density lipoproteins), nucleic acids, carbohydrates, and small molecule toxins. Recombinant polypeptides resulting from translation of mRNA can be engineered for localization within a cell, such as within a specific compartment such as the nucleus, or can be engineered for secretion by the cell or translocation to the cell's plasma membrane.

In some embodiments, contacting cells with mRNA-containing lipid nanoparticles (eg, empty LNPs or loaded LNPs) can reduce the cellular innate immune response to exogenous nucleic acids. A cell can be contacted with a first lipid nanoparticle (eg, empty LNP or loaded LNP) comprising a first amount of a first exogenous mRNA comprising a translatable region, and the response of the cell to the first exogenous mRNA can be determined. Levels of innate immune responses to sex mRNAs. Subsequently, the cell can be contacted with a second composition comprising a second amount of the first exogenous mRNA, the second amount being a lower amount of the first exogenous mRNA compared to the first amount. Alternatively, the second composition may comprise a first amount of a second exogenous mRNA that is different from the first exogenous mRNA. The step of contacting the cells with the first and second compositions can be repeated one or more times. Additionally, the efficiency of polypeptide production (eg, translation) in cells can optionally be determined, and the cells can be repeatedly recontacted with the first and/or second compositions until the desired protein production efficiency is achieved.

Methods of delivering therapeutic agents to cells and organs

The present disclosure provides methods of delivering therapeutic and/or prophylactic agents to mammalian cells or organs. Delivering a therapeutic and/or prophylactic agent to a cell comprises administering to a subject a lipid nanoparticle (eg, empty LNP or loaded LNP) comprising the therapeutic and/or prophylactic agent, wherein administration of the composition Including contacting the cells with the composition. For example, proteins, cytotoxic agents, radioactive ions, chemotherapeutic agents, or nucleic acids (such as RNA, eg, mRNA) can be delivered to cells or organs. Where the therapeutic and/or prophylactic agent is mRNA, upon contact of the cell with the nanoparticle composition, the translatable mRNA can be translated in the cell to produce the polypeptide of interest. However, substantially untranslatable mRNA can also be delivered to cells. Substantially untranslatable mRNA can be used as a vaccine and/or can block the translational components of the cell to reduce the expression of other substances in the cell.

In some embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can target specific types or classes of cells (eg, cells of specific organs or systems thereof). For example, lipid nanoparticles (eg, empty LNPs or loaded LNPs) containing therapeutic and/or prophylactic agents of interest can be specifically delivered to mammalian liver, kidney, spleen, or lung. Specific delivery to specific classes of cells, organs or systems, or groups thereof, implies a higher proportion of lipid nanoparticles (e.g., loaded with LNPs) containing therapeutic and/or prophylactic agents relative to other destinations is delivered to the destination of interest (eg, organization). In some embodiments, specific delivery of an mRNA-containing LNP-loaded can result in cells of a targeted destination (eg, a tissue of interest, such as the liver) compared to cells of another destination (eg, the spleen) The mRNA expression increased by greater than 2-fold, 5-fold, 10-fold, 15-fold or 20-fold. In some embodiments, the tissue of interest is selected from the group consisting of liver, kidney, lung, spleen, and tumor tissue (eg, by intratumoral injection).

In some embodiments, specific delivery of mRNA contained in a loaded LNP of the present disclosure (ie, a lipid nanoparticle formulated with a compound of the present disclosure) can result in a That is, there are no formulas (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) lipids) compared to , mRNA expression increased by greater than 2-fold, 5-fold, 10-fold, 15-fold or 20-fold.

As another example of targeted or specific delivery, mRNA encoding protein binding partners (eg, antibodies or functional fragments thereof, scaffold proteins or peptides) or receptors on the cell surface can be included in nanoparticle compositions. mRNA can additionally or alternatively be used to direct the synthesis and extracellular localization of lipids, carbohydrates or other biological moieties. Alternatively, other therapeutic and/or prophylactic agents or components (eg, lipids or ligands) in lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be based on their ability to respond to specific receptors (eg, low-density lipoproteins). receptors) so that lipid nanoparticles (eg, empty LNPs or loaded LNPs) can more readily interact with target cell populations that include these receptors. For example, ligands can include, but are not limited to, members of a given binding pair, antibodies, monoclonal antibodies, Fv fragments, single chain Fv (scFv) fragments, Fab' fragments, F(ab')2 fragments, single domain antibodies, camelid Antibodies and fragments thereof, humanized antibodies and fragments thereof, and multivalent versions thereof; multivalent binding reagents, including monospecific or bispecific antibodies such as disulfide stabilized Fv fragments, scFv concatemers, diabodies , tri- or tetrabodies; and aptamers, receptors, and fusion proteins.

In some embodiments, the ligand may be a surface-bound antibody, which may allow tuning of cellular targeting specificity. This is especially useful because highly specific antibodies can be raised against epitopes of interest at the desired targeting site. In some embodiments, multiple antibodies are expressed on the cell surface, and each antibody may have different specificities for a desired target. Such methods can increase the affinity and specificity of targeted interactions.

For example, one skilled in the art of biology can select ligands based on the desired cellular localization or function.

Targeted cells can include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells , cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.

In some embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can target hepatocytes. Apolipoproteins such as apolipoprotein E (apoE) have been shown to associate with neutral or near-neutral lipid-containing lipid nanoparticles (eg, empty LNPs or loaded LNPs) in vivo, and are known to Lipoproteins associate with receptors found on the surface of liver cells, such as the low density lipoprotein receptor (LDLR). Thus, a nanoparticle composition comprising a lipid component with a neutral or near-neutral charge administered to a subject can acquire apoE in the subject, and subsequently therapeutic and/or prophylactic agents (e.g., , RNA) in a targeted manner to LDLR-containing hepatocytes.

Methods of Treating Diseases and Conditions

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be used to treat diseases, disorders or conditions. In particular, such compositions can be used to treat diseases, disorders or conditions characterized by missing or abnormal protein or polypeptide activity. For example, lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising mRNA encoding a deleted or abnormal polypeptide can be administered or delivered to cells. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by the lack of or abnormal activity of the polypeptide. Because translation can occur rapidly, the methods and compositions can be used to treat acute diseases, disorders or conditions such as sepsis, stroke and myocardial infarction. Therapeutic and/or prophylactic agents contained in lipid nanoparticles (eg, empty LNPs or loaded LNPs) can also alter the transcription rate of a given substance, thereby affecting gene expression.

Diseases, disorders and/or conditions characterized by dysfunctional or abnormal protein or polypeptide activity to which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer and proliferative diseases, Genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renovascular diseases, and metabolic diseases. A variety of diseases, disorders and/or conditions can be characterized by a loss of protein activity (or a substantial reduction such that proper protein function cannot occur). Such proteins may not exist, or they may be essentially nonfunctional. The present disclosure provides a method of treating such diseases, disorders and/or conditions in a subject by administering lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising RNA and lipids component, the lipid component comprises according to formula (A), (A1), (A2), (A3), (B), (I), (I'), (I-X), (I-Y), ( I-Ya) lipids of (IA), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb) , phospholipids (optionally unsaturated), PEG lipids, and structural lipids, wherein the RNA may be an mRNA encoding a polypeptide that antagonizes or otherwise overcomes the activity of an abnormal protein present in a subject's cells.

The present disclosure provides methods comprising administering lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising one or more therapeutic and/or prophylactic agents, and pharmaceutical compositions comprising the lipid nanoparticles. For features and embodiments of the present disclosure, the terms therapeutic agent and prophylactic agent are used interchangeably herein. Therapeutic compositions or imaging, diagnostic or prophylactic compositions thereof can be administered to a subject using any reasonable amount and by any route of administration effective to prevent, treat, diagnose or image a disease, disorder and/or condition and/or for any other purpose. The indicated amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of administration; the particular composition; the mode of administration, and the like. Compositions according to the present disclosure can be formulated in unit dosage form for ease of administration and uniformity of dosage. It is to be understood, however, that the total daily dosage of the compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. The indicated therapeutically effective, prophylactically effective, or other appropriate dosage level for any particular patient (eg, for imaging) will depend on a variety of factors, including the severity and nature of the condition being treated (if present); one or more therapeutic and/or prophylactic agents; the specified composition employed; the patient's age, weight, general health, gender and diet; the administration time, route of administration and excretion rate of the specified pharmaceutical composition employed; treatment duration time; drugs used in combination or concomitantly with the prescribed pharmaceutical composition employed; and similar factors well known in the medical arts.

The loaded LNP can be administered by any route. In some embodiments, one or more LNP-loaded compositions described herein, including prophylactic, diagnostic or imaging compositions, are administered by one or more of a variety of routes including: oral, Intravenous, intramuscular, intraarterial, subcutaneous, transdermal or intradermal, interdermal, intraperitoneal, mucosal, nasal, intratumoral, intranasal; administration by inhalation; as oral spray and/or powder, nasal spray and/or by aerosol, and/or by portal vein catheter. In some embodiments, the composition may be administered intravenously, intramuscularly, intradermally, intraarterally, intratumorally, subcutaneously or by any other parenteral route of administration or by inhalation. However, the present disclosure contemplates delivery or administration of the compositions described herein by any suitable route, given possible advances in drug delivery science. In general, the most appropriate route of administration will depend on a variety of factors, including the nature of the loaded LNP comprising one or more therapeutic and/or prophylactic agents (eg, its exposure to various bodily environments such as the bloodstream and the gastrointestinal tract). stability in the tract), the condition of the patient (eg, whether the patient can tolerate a particular route of administration), and the like.

In certain embodiments, compositions according to the present disclosure may be sufficient to deliver about 0.0001 mg/kg to about 10 mg/kg, about 0.001 mg/kg to about 10 mg/kg, about 0.005 mg/kg to about 0.0001 mg/kg in a given dose 10 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg, about 2 mg/kg kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 0.0001 mg/kg to about 5 mg/kg, about 0.001 mg/kg to about 5 mg/kg, about 0.005 mg/kg to about 5 mg/kg, About 0.01 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 5 mg/kg, about 2 mg/kg to about 5 mg /kg, about 0.0001 mg/kg to about 2.5 mg/kg, about 0.001 mg/kg to about 2.5 mg/kg, about 0.005 mg/kg to about 2.5 mg/kg, about 0.01 mg/kg to about 2.5 mg/kg , about 0.05 mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 2.5 mg/kg, about 1 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 0.0001 mg /kg to about 1 mg/kg, about 0.001 mg/kg to about 1 mg/kg, about 0.005 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.05 mg/kg to about 1 mg/kg kg, about 0.1 mg/kg to about 1 mg/kg, about 0.0001 mg/kg to about 0.25 mg/kg, about 0.001 mg/kg to about 0.25 mg/kg, about 0.005 mg/kg to about 0.25 mg/kg, about Dose levels of 0.01 mg/kg to about 0.25 mg/kg, about 0.05 mg/kg to about 0.25 mg/kg, or about 0.1 mg/kg to about 0.25 mg/kg therapeutic and/or prophylactic (eg, mRNA) to be administered, wherein a dose of 1 mg/kg (mpk) provides 1 mg of the therapeutic and/or prophylactic agent per 1 kg of subject body weight. In some embodiments, a dose of about 0.001 mg/kg to about 10 mg/kg of the LNP-loaded therapeutic and/or prophylactic agent can be administered. In other embodiments, doses of about 0.005 mg/kg to about 2.5 mg/kg of therapeutic and/or prophylactic agents can be administered. In certain embodiments, a dose of about 0.1 mg/kg to about 1 mg/kg may be administered. In other embodiments, a dose of about 0.05 mg/kg to about 0.25 mg/kg may be administered. Doses may be administered one or more times per day in the same or different amounts to achieve the desired level of mRNA expression and/or therapeutic, diagnostic, prophylactic or imaging effect. The desired dose is delivered, for example, three times a day, twice a day, once a day, every other day, every three days, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose may be administered using multiple administrations (eg, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve administration, thirteen, fourteen or more administrations). In some embodiments, a single dose may be administered, eg, before or after a surgical procedure or in the case of an acute disease, disorder or condition.

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising one or more therapeutic and/or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" is not intended to indicate that the agents must be administered at the same time and/or formulated for delivery together, although such delivery methods are within the scope of this disclosure. For example, one or more lipid nanoparticles (eg, empty LNPs or loaded LNPs) comprising one or more different therapeutic and/or prophylactic agents can be administered in combination. The compositions can be administered concurrently with, before, or after one or more other desired therapeutic agents or medical procedures. In general, each agent will be administered at the dose and/or schedule established for that agent. In some embodiments, the present disclosure contemplates delivering a composition or imaging, diagnostic or prophylactic composition thereof with an agent that improves its bioavailability, reduces and/or improves its metabolism, inhibits its excretion, and/or improves its distribution in the body The combination.

It will also be appreciated that therapeutic, prophylactic, diagnostic or imaging active agents used in combination may be administered together in a single composition or administered separately in different compositions. In general, it is contemplated that the agents used in combination will be used at levels that do not exceed the levels at which they are used individually. In some embodiments, the levels used in combination may be lower than those used alone.

The particular combination of therapies (therapeutic agents or procedures) used in a combination regimen will take into account the compatibility of the desired therapeutic agents and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapy employed may achieve the desired effect for the same condition (eg, a composition useful in the treatment of cancer may be administered concurrently with the chemotherapeutic agent), or the therapy may achieve a different effect (eg, to control any adverse effects, such as infusion-related reactions).

Lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be used in combination with agents that increase the effectiveness and/or therapeutic window of the composition. Such agents may be, for example, anti-inflammatory compounds, steroids (eg, corticosteroids), statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators (GRMs), or antihistamines. In some embodiments, lipid nanoparticles (eg, empty LNPs or loaded LNPs) can be used in combination with dexamethasone, methotrexate, acetaminophen, H1 receptor blockers, or H2 receptor blockers . In some embodiments, a method of treating a subject in need thereof or delivering a therapeutic and/or prophylactic agent to a subject (eg, a mammal) can include, prior to administering the nanoparticle composition, using one or more The subject is pretreated with a dose. For example, a useful amount (eg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, or any other available amount) of dexamethasone, methotrexate, acetaminophen, H1 can be used Subjects were pretreated with a receptor blocker or an H2 receptor blocker. Pre-treatment can be administered 24 hours or less (eg, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hours, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes) and can be performed once, twice, or more, eg, in escalating doses.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein in accordance with the present disclosure. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

In the claims, articles such as "a", "an" and "the" may mean one or the other, unless indicated to the contrary or otherwise clear from the context. More than one/species. Unless indicated to the contrary or otherwise apparent from the context, one, more than one or all of the group members are considered to be present if present in, used in, or otherwise related to a given product or process It is sufficient for a claim or description to include "or" between one or more members of the group. The present disclosure includes embodiments in which only one member of the group is present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which more than one or all of the group members are present in, used in, or otherwise related to a given product or process. As used herein, the expressions "one or more of A, B, or C," "one or more of A, B, or C," "one or more of A, B, and C," unless otherwise specified, "One or more of A, B, and C," "selected from A, B, and C," "selected from the group consisting of A, B, and C," and the like are used interchangeably and all refer to the term "selected from the group consisting of A, B, and C." and/or selected from the group consisting of C, ie one or more A's, one or more B's, one or more C's, or any combination thereof.

It should also be noted that the term "comprising" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting essentially of" and "consisting of" are hereby also encompassed and disclosed. Throughout the specification, where compositions are described as having, comprising or comprising the specified components, it is contemplated that the compositions also consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, comprising or comprising specified process steps, the process also consists essentially of or consists of the recited process steps. Additionally, it should be understood that the order of steps or order in which certain actions are performed is immaterial so long as the invention remains operable. Furthermore, two or more steps or actions may be performed simultaneously.

Where a range is given, the endpoints are included. Furthermore, it should be understood that the values expressed as ranges may assume any specified value within the range in various embodiments of the present disclosure unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art or sub-ranges up to the tenth of the unit of the lower limit of the stated range, unless the context clearly dictates otherwise.

The synthetic procedures of the present disclosure can tolerate a wide variety of functional groups and thus can use a variety of substituted starting materials. These procedures generally provide the desired final compound at or near the end of the overall procedure, although in some cases it may be desirable to further convert the compound to its pharmaceutically acceptable salt.

The compounds of the present disclosure can be obtained by employing standard synthetic methods and procedures known to or apparent to those skilled in the art from the teachings herein using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates Bodies are prepared in a variety of ways. Standard synthetic methods and procedures for preparing organic molecules and functional group transformations and manipulations can be obtained from the relevant scientific literature or from standard textbooks in the field. While not limited to any one or several sources, classic textbooks such as Smith, M.B., March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition, are incorporated herein by reference, John Wiley & Sons: New York, 2001; Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999; R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); L. Fieser and M. . Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), are available and are of skill in the art A recognized organic synthesis reference textbook known to personnel. The following descriptions of synthetic methods are designed to illustrate, but not to limit, general procedures for the preparation of compounds of the present disclosure.

Compounds of the present disclosure having any of the formulae described herein can be prepared according to the procedures illustrated in Schemes 1, 2, and 3 below from commercially available starting materials or starting materials that can be prepared using literature procedures . The variables in the protocol (eg, R ¹ , R ² and R ³ etc. are as defined herein). One of ordinary skill in the art will note that the order of certain steps, such as the introduction and removal of protecting groups, may be altered during the reaction procedures and synthetic schemes described herein.

One of ordinary skill in the art will recognize that it may be desirable to protect certain groups from reaction conditions through the use of protecting groups. Protecting groups can also be used to distinguish similar functional groups in a molecule. A list of protecting groups and methods of introducing and removing these groups can be found in Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons: New York, 1999.

Preferred protecting groups include, but are not limited to:

For hydroxyl moieties: TBS, benzyl, THP, Ac;

For carboxylic acids: benzyl ester, methyl ester, ethyl ester, allyl ester;

For amines: Fmoc, Cbz, BOC, DMB, Ac, Bn, Tr, Ts, trifluoroacetyl, phthalimide, benzylideneamine;

For diols: Ac(x2)TBS(x2), or acetonate when put together;

For thiols: Ac;

For benzimidazole: SEM, benzyl, PMB, DMB;

For aldehydes: dialkyl acetals such as dimethoxyacetal or diethylacetyl.

In the reaction schemes described herein, various stereoisomers can be produced. When no specific stereoisomer is indicated, it is understood to mean all possible stereoisomers that can result from the reaction. One of ordinary skill in the art will recognize that the reactions can be optimized to give preference to one isomer, or new schemes can be devised to produce a single isomer. If a mixture is produced, the isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC.

plan 1

As shown in Scheme 1 above, 8-bromooctanoic acid is reacted with alcohol a1 (eg, heptadecan-9-ol) to give ester b1 (eg, heptadecan-9-yl 8-bromooctanoate). Step 1 can be carried out in an organic solvent ( For example, dichloromethane). Step 1 can be performed at room temperature for 18 hours. Next, ester b1 is reacted with 2-aminoethane-1-ol to give amine c1 (eg, 8-((2-hydroxyethyl)amino)octanoic acid heptadecan-9-yl ester). Step 2 can be carried out in ethanol, for example, at a temperature of about 60°C. The amine c1 is then reacted with a bromoalkyl R1 ^- Br (eg, 1-bromotetradecane) to give compound d1 (eg, 8-((2-hydroxyethyl)(tetradecyl)amino)octanoic acid ten heptan-9-yl ester). Step 3 can be carried out in ethanol in the presence of N,N-diisopropylethylamine.

Scenario 2

As shown in Scheme 2 above, acid a2 ( ^x3 is an integer between 1 and 7; eg, 8-bromooctanoic acid) is reacted with alcohol b2 (eg, nonan-1-ol) to give ester c2 (eg, , nonyl 8-bromooctanoate). Step 1 can be carried out in an organic solvent ( For example, dichloromethane). Alcohols e2 (eg, heptadecan-9-ol) can be obtained by reacting an aldehyde d2 (eg, nonanal) with a Grignard reagent ^R3 - _MgX (eg, _nC8H17MgBr ) via Step 2. Next, 8-bromooctanoic acid is reacted with alcohol e2 (eg, heptadecan-9-ol) to give ester f2 (eg, 8-bromooctanoic acid heptadecan-9-yl ester). Step 3 can be carried out in an organic solvent ( For example, dichloromethane). Next, ester f2 is reacted with 2-aminoethane-1-ol to give amine g2 (eg, heptadecan-9-yl 8-((2-hydroxyethyl)amino)octanoate). Step 4 can be carried out in ethanol in the presence of i _- Pr2EtN. Then, amine g2 is reacted with ester c2 (eg, nonyl 8-bromooctanoate) to give compound h2 (eg, 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl) ) amino) heptadecan-9-yl octanoate). Step 5 can be performed in a base such as an inorganic base (eg, K ₂ CO ₃ ) or a non-nucleophilic organic base (eg, i-Pr ₂ ) at, for example, elevated temperature (such as at about 70°C-90°C, eg, about 80°C). EtN)) and a catalyst (eg, an iodide such as KI or NaI) in an organic solvent (eg, a mixture of CPME and MeCN).

Scenario 3

As shown in Scheme 3 above, haloalkanol ( ^x3 is an integer between 1 and 12, eg, 6-bromohexane-1-ol) and starting material a3 ( ^x2 is between 1 and 6) an integer between, eg, 4-(hexyloxy)-4-oxobutyric acid) to give the halodiester b3 (eg, 6-bromohexylhexyl succinate). Compound a3 can pass through alcohol (for example, hexane-1-ol) and acid anhydride (for example, succinic anhydride, dihydro-2H-pyran-2,6(3H)-dione, 3-(tert-butoxy)- 3-oxopropionic acid, 4-(tert-butoxy)-3-methyl-4-oxobutyric acid or 4-(tert-butoxy)-2-methyl-4-oxobutyric acid) reaction to obtain. Step 1 can be carried out in an organic solvent ( For example, dichloromethane). Next, the halogenated diester b3 and the amine c3 (x ⁴ is an integer between 5 and 13, x ⁵ is an integer between 1 and 5, eg, 8-((2-hydroxyethyl) amino) heptadecan-9-yl octanoate) to give product d3. Step 2 can be carried out at elevated temperature (eg, about 90°C) in a base (such as an inorganic base (eg, K2CO3 ₎ and a catalyst (eg, an _iodide such as KI) and an ether solvent (eg, cyclopentyl methyl ether) ) in an organic solvent (eg, a mixture of CPME and MeCN).

One of ordinary skill in the art will recognize that in the above schemes, the order of certain steps may be interchanged.

In certain aspects, the present disclosure also includes the synthesis of formulae (1-1), (2-1), (I-a), (A), (B), (A-1), (A-2), (A- 3), (IA), (IB), (B-1), (B-2), (B-3), (A-a), (A-a1), (A-a2), (A-a3) Compounds of any of , (A-b), (A-b1), (A-b2), (A-b3), (A-c) and (B-c) and methods for synthesizing intermediates of said compounds.

与R¹-Br反应以得到本公开的化合物，其中各变量如本文所定义。例如，m是5、6、7、8或9，优选5、7或9。例如，R⁵、R⁶和R⁷中的每一个是H。例如，M是-C(O)O-或-OC(O)-。例如，R⁴是未取代的C_1-3烷基或-(CH₂)_nQ，其中n为2、3或4并且Q为OH、-NHC(S)N(R)₂、-NHC(O)N(R)₂、-N(R)C(O)R或-N(R)S(O)₂R。例如，式(X2)的化合物与R¹-Br的反应在碱(诸如无机碱(例如，K₂CO₃)或非亲核性有机碱(例如，i-Pr₂EtN))的存在下进行。例如，所述反应是在无机碱(例如，K₂CO₃)和催化剂(例如，碘化物，诸如KI或NaI)的存在下进行。例如，所述反应是在高温(例如，在约50℃-100℃、70℃-90℃或约80℃)下进行。In some embodiments, methods of synthesizing the compounds of the present disclosure include making a compound of formula (X2):

Reaction with R1 ^- Br to give compounds of the present disclosure, wherein the variables are as defined herein. For example, m is 5, 6, 7, 8 or 9, preferably 5, 7 or 9. For example, each of R ⁵ , R ⁶ and R ⁷ is H. For example, M is -C(O)O- or -OC(O)-. For example, R4 is unsubstituted _C1-3 alkyl or -( _CH2 )nQ, where _n is 2, 3 or ⁴ and Q is OH, -NHC(S)N(R) ₂ , -NHC( O)N(R) ₂ , -N(R)C(O)R or -N(R)S(O ₎ 2R. For example, the reaction of a compound of formula (X2) with R ¹ -Br is carried out in the presence of a base such as an inorganic base (eg, K ₂ CO ₃ ) or a non-nucleophilic organic base (eg, i-Pr ₂ EtN) . For example, the reaction is carried out in the presence of an inorganic base (eg, K2CO3 ₎ and a catalyst (eg, an _iodide such as KI or NaI). For example, the reaction is carried out at elevated temperature (eg, at about 50°C-100°C, 70°C-90°C, or about 80°C).

The method can also include making the compound of formula (X1):

与R⁴NH₂反应以得到式(X2)的化合物，其中各变量如本文所定义。

Reaction with R4NH2 to give _compounds of ^formula (X2), wherein the variables are as defined herein.

其中各变量如本文所定义。例如，中间体包括8-溴辛酸十七烷-9-基酯和8-((2-羟乙基)氨基)辛酸十七烷-9-基酯及其形态形式(例如，结晶形式)。In some embodiments, intermediates include those of any of formulae (X1) and (X2):

where the variables are as defined herein. For example, intermediates include heptadec-9-yl 8-bromooctanoate and heptadec-9-yl 8-((2-hydroxyethyl)amino)octanoate and morphological forms (eg, crystalline forms) thereof.

Furthermore, it is to be understood that any particular embodiment of the present disclosure that is within the scope of the prior art may be expressly excluded from any claim or claims. Because such embodiments are believed to be known to those of ordinary skill in the art, they may be excluded even if not explicitly stated herein.

All cited sources, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of a conflict between a cited source and the expression in the present application, the expression in the present application shall control.

Example

Example 1: Synthesis of the compounds of Table 1

A. General Considerations

Unless otherwise stated, all solvents and reagents used were obtained commercially and used as received. < ¹ >HNMR spectra were recorded in _CDCl3 at 300K using a Bruker Ultrashield 300MHz instrument. Chemical shifts are reported in parts per million (ppm) relative to ¹ H TMS (0.00). Silica gel chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using an ISCO RediSep Rf Gold flash column (particle size: 20-40 microns). Reversed-phase chromatography was performed on an ISCO CombiFlash Rf+Lumen instrument using a RediSep Rf Gold C18 high performance column. Using a WatersAcquity UPLC instrument with DAD and ELSD and a ZORBAX Rapid Resolution High Definition (RRHD) SB-C18 LC column (2.1 mm, 50 mm, 1.8 μm) and 0.1% TFA in water at 1.2 mL/min over 5 minutes Gradient from 65% to 100% acetonitrile, all final compounds were determined to be greater than 85% pure via analysis by reverse phase UPLC-MS (retention time, RT, in minutes). The injection volume was 5 μL and the column temperature was 80°C. Detection was performed in positive ionization mode based on electrospray ionization (ESI) using a Waters SQD mass spectrometer (Milford, MA, USA) and an evaporative light scattering detector.

The procedure described below can be used to synthesize the compounds of Table 1.

The following abbreviations are used in this article:

THF: Tetrahydrofuran

MeCN: Acetonitrile

LAH: Lithium Aluminum Hydride

DCM: dichloromethane

DMAP: 4-Dimethylaminopyridine

LDA: Lithium Diisopropylamide

rt: room temperature

DME: 1,2-Dimethoxyethane

n-BuLi: n-butyllithium

CPME: cyclopentyl methyl ether

i-Pr ₂ EtN: N,N-diisopropylethylamine

AA. Compound 6: Heptadecan-9-yl 8-((2-hydroxyethyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoate

UPLC/ELSD: RT=3.12 min. MS ( _ES ) for _C43H85NO6 : m/z (MH ⁺ ) _713.191

¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.89 (p, 1H); 4.17 (t, 4H); 3.56 (bm, 2H); 2.75-2.36 (m, 6H); 2.30 (t, 2H); 1.77 -1.20 (m, 61H); 0.90 (m, 9H).

AB. Compound 25: 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-((( Nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9-yl ester(4-nitrophenyl)6-bromohexyl carbonate

In a round bottom flask equipped with a magnetic stirrer, 4-nitrophenyl chloroformate (9.35 g, 46.39 mmol) was added in one portion in DCM (100 mL) at room temperature to give 6-bromo-1- in hexanol (7 g, 38.658 mmol). The reaction was kept under _N2 and pyridine (4.69 mL, 57.988 mmol) was added dropwise over 10 minutes. Then stirring was continued for 16 hours at room temperature. The reaction was diluted with water and DCM. The organic layer was separated and the aqueous layer was washed with DCM. The combined organic layers were washed with brine, dried _{over Na2SO4} and evaporated in vacuo. The residue was purified by silica gel chromatography (0%-100% ethyl acetate in hexanes) to give (4-nitrophenyl)6-bromohexyl carbonate (10.3 g, 38.7 mmol, 77% ). ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 8.29 (d, 2H); 7.43 (d, 2H); 4.33 (t, 2H); 3.43 (t, 2H); 2.02-1.69 (m, 4H); 1.53 (m, 4H).

6-Bromohexyl nonyl carbonate

Non-1-ol (8.334 g, 57.774 mmol) was added dropwise to 4-nitrobenzene in DCM ( ₁₅ mL) at room temperature under N in a round bottom flask equipped with a magnetic stirrer in a solution of 6-bromohexyl carbonate (5 g, 14.443 mmol). The reaction was kept under _N2 and pyridine (1.46 mL, 18.054 mmol) was added dropwise over 10 minutes. Then 4-dimethylaminopyridine (0.353 g, 2.889 mmol) was added in one portion. The reaction mixture was stirred at room temperature for 16 hours, then diluted with water and DCM. The organic layer was separated and the aqueous layer was washed with DCM. The combined organic layers were washed with brine, dried _{over Na2SO4} and evaporated in vacuo. The residue was purified by silica gel chromatography (0%-100% ethyl acetate in hexanes) to give 6-bromohexyl nonyl carbonate (1.3 g, 3.7 mmol, 26%). ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.15 (m, 4H); 3.43 (t, 2H); 1.89 (m, 2H); 1.71 (m, 4H); 1.58-1.18 (m, 16H); 0.90 (m, 3H).

Heptadecan-9-yl 8-((3-((tert-butoxycarbonyl)amino)propyl)amino)octanoate

A solution of heptadec-9-yl 8-bromooctanoate (69.2 g, 0.15 mol) and tert-butyl (3-aminopropyl)carbamate (130.6 g, 0.75 mol) in 500 mL of ethanol was heated to 65 °C overnight. The reaction mixture was concentrated and the crude product was purified by flash column chromatography ( _SiO2 : methanol/dichloromethane 0%-20%) to give 8-((3-((tert-butoxycarbonyl) as a pale yellow oil Amino))propyl)amino)heptadecan-9-yl octanoate (62 g, 74%). MS (CI) for _C33H66N2O4 : m/z (MH^&lt;+ _{& gt ;} ) 555.5. ¹ H NMR (300 MHz, CDCl ₃ ): δppm 5.15 (bs, 1H); 4.85 (quint., 1H, J=6.0 Hz); 3.17 (m, 2H); 2.65 (t, 2H, J=6.6 Hz); 2.56(t,2H,J=6.8Hz);2.26(t,2H,J=7.6Hz);1.68-1.56(m,6H);1.46(m,5H);1.43(s,9H);1.24(m , 30H); 0.86 (t, 6H, J=6.6Hz).

8-((3-((tert-butoxycarbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9-yl ester

To nonyl 6-bromohexyl carbonate (0.633 g, 1.802 mmol) and 8-((3-((tert-butoxycarbonyl)amino) in 40 mL of a 1:1 mixture of cyclopentyl methyl ether and acetonitrile To a solution of propyl)amino)octanoic acid heptadecan-9-yl ester (1 g, 1.802 mmol) was added potassium carbonate (0.996 g, 7.208 mmol) and potassium iodide (0.329 g, 1.982 mmol). The reaction was heated to 77°C and stirred for 16 hours. The solution was cooled, filtered, and the volatiles were evaporated in vacuo. The residue was purified by silica gel chromatography (0%-100% (20% MeOH, 80% DCM, 1% _NH4OH ) in DCM) to give 8-((3-(((tert-butoxy) Carbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (0.32 g, 0.39 mmol, 22%). UPLC/ELSD: RT=3.02 min. MS (ES) for _C49H96N2O7 : m/z (MH&lt _; ⁺ &gt;) _826.300 . ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 5.65 (bm, 1H); 4.88 (m, 1H); 4.14 (t, 4H); 3.19 (bm, 2H); 3.00 (bm, 1H); 2.52-2.22 (m, 7H); 1.76-1.17 (m, 71H); 0.90 (m, 9H).

8-((3-Aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9-yl ester

To 8-((3-((tert-butoxycarbonyl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9 in 3 mL of DCM Trifluoroacetic acid (0.6 mL, 7.8 mmol) was added to a solution of -yl ester (320 mg, 0.39 mmol), and the solution was stirred at room temperature for 16 hours. The reaction was quenched by addition of saturated sodium bicarbonate at 0 °C. The organic layer was washed with saturated sodium bicarbonate solution and brine. After drying over anhydrous sodium sulfate, the solvent was removed in vacuo to give 8-((3-aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9- base ester (283 mg, quantitative). UPLC/ELSD: RT=2.39 min. MS (ES) for _C44H88N2O5 : m/z (MH&lt _; ⁺ &gt;) _726.142 . ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.87 (m, 1H); 4.14 (m, 4H); 3.21-2.97 (m, 4H); 2.78 (m, 4H); 2.30 (m, 2H); 2.04 (m, 2H); 1.80-1.17 (m, 60H); 0.90 (m, 9H).

3-Methoxy-4-(methylamino)cyclobut-ene-1,2-dione

To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione (1 g, 7 mmol) in 100 mL diethyl ether was added 2M methane in THF (3.8 mL, 7.6 mmol) base amine solution and a precipitate formed almost immediately. The mixture was stirred at room temperature for 24 hours, then filtered, and the filtered solids were washed with diethyl ether and air dried. The filtered solid was dissolved in hot EtOAc, filtered, and the filtrate was allowed to cool to room temperature and then to 0°C to give a precipitate. It was isolated by filtration, washed with cold EtOAc, air dried, then vacuum dried to give 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione as a white solid (0.70 g, 5 mmol, 73%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ: ppm 8.50 (br.d, 1H, J=69 Hz); 4.27 (s, 3H); 3.02 (sdd, 3H, J=42 Hz, 4.5 Hz).

8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-(nonyloxy)carbonyl)oxy yl)hexyl)amino)heptadecan-9-yl octanoate

To 8-((3-aminopropyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecan-9- in THF (2 mL) and water (0.4 mL) 3-methoxy-4-(methylamino)cyclobut-3-ene-1,2-dione (0.06 g, 0.41 mmol) was added to a solution of the base ester (0.2 g, 0.28 mmol) and the reaction was heated to 67°C and stirred for 20 hours. The reaction was allowed to cool to room temperature during which time the clear solution became a cloudy mixture. Anhydrous _MgSO4 was added, the reaction was filtered and the filtrate was concentrated. The residue was purified by silica gel chromatography (0%-100% (20% MeOH, 80% DCM, 1% _NH4OH ) in DCM) to give 8-((3-((2-(methyl (ylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-(((nonyloxy)carbonyl)oxy)hexyl)amino)octanoic acid heptadecane -9-ylester (120 mg, 0.14 mmol, 50%). UPLC/ELSD: RT=2.81 min. MS (ES) for _C49H91N3O7 : _m /z (MH&lt _; ⁺ &gt;) _835.058 . ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.88 (p, 1H); 4.14 (m, 4H); 3.74 (m, 2H); 3.40-2.90 (m, 9H); 2.31 (m, 2H); 2.12 (m, 2H); 1.80-1.20 (m, 62H); 0.90 (m, 9H).

AC. Compound 64: Heptadecan-9-yl 8-((2-hydroxyethyl)(6-((((3-pentyloctyl)oxy)carbonyl)oxy)hexyl)amino)octanoate

UPLC/ELSD: RT=3.04 min. MS ( _ES ) for _C47H93NO6 : m/z (MH ⁺ ) _769.313

¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.89 (p, 1H); 4.14 (m, 4H); 3.54 (bm, 2H); 2.66-2.37 (m, 6H); 2.30 (m, 2H); 1.77 -1.17 (m, 66H); 0.91 (m, 12H).

AD. Compound 65: 8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(3-((2-(methylamino)-3,4-dioxo) Cyclobut-1-en-1-yl)amino)propyl)amino)octanoic acid heptadecan-9-yl ester 3-hexylnonan-2-acid ethyl ester

To a suspension of sodium hydride (4.697 g, 117.4 mmol) in THF (294 mL) was added triethyl phosphonoacetate (26.33 g, 117.4 mmol) dropwise over 20 min and the mixture was stirred at room temperature until gassed Escape stopped (about 30 minutes). The reaction mixture was cooled to 0 °C and 7-tridecone (10 g, 58.7 mmol) was added. The reaction was gradually warmed to room temperature, then heated to reflux and stirred overnight. The reaction was quenched with saturated aqueous sodium bicarbonate. The aqueous phase was extracted with ether, and the organic extracts were washed with brine, dried over _MgSO4 and concentrated. The crude material was purified by silica gel chromatography (0%-20% EtOAc:hexanes) to give ethyl 3-hexylnon-2-acid (6.7 g, 27.9 mmol, 47.5%) as a clear oil. ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 5.63 (s, 1H); 4.15 (q, 2H); 2.61 (t, 2H); 2.15 (t, 2H); 1.53-1.20 (m, 19H); 0.91 (m, 6H).

Ethyl 3-hexylnonanoate

To a flask containing a slurry of Pearlmans catalyst (0.73 g, 5.2 mmol) in ethanol (20 mL) was added ethyl 3-hexylnon-2-enoate (6.975 g) in ethanol (5 mL) under _N2 , 25.9 mmol) solution. The reaction was stirred under _H2 (balloon) for 16 hours. The reaction was filtered through a plug of celite, and the filtrate was evaporated in vacuo to give ethyl 3-hexylnonanoate (6.7 g, 24.7 mmol, 95%). The residue was used in the next step without further purification. ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.16 (q, 2H); 2.23 (d, 2H); 1.86 (bs, 1H); 1.28 (m, 23H); 0.90 (m, 6H).

3-Hexylnonan-1-ol

To a solution of lithium aluminum hydride in THF (49.5 mL of a 1M solution in THF, 49.5 mmol) was added a solution of ethyl 3-hexylnonanoate (6.7 g, 24.7 mmol) in THF (20 mL). The reaction was continued at room temperature for 16 hours. The reaction was quenched with saturated sodium sulfate decahydrate solution. The white solid was removed by filtration through a plug of celite, and the filtrate was evaporated in vacuo. The residue was purified by flash chromatography (ISCO) with 0%-100% ethyl acetate in hexanes to obtain 3-hexylnon-1-ol (5.62 g, 24.6 mmol, 99%). ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 3.69 (t, 2H); 1.61-1.19 (m, 24H); 0.91 (m, 6H).

8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(3-((2-(methylamino)-3,4-dioxocyclobutane-1) -En-1-yl)amino)propyl)amino)octanoic acid heptadecan-9-yl ester

Compound 65 was prepared analogously to compound 25 using 3-hexylnonan-1-ol in place of nonan-1-ol. UPLC/ELSD: RT=3.13 min. MS (ES) for _C55H103N3O7 : _m /z (MH&lt _; ⁺ &gt;) _919.429 . ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.87 (p, 1H); 4.17 (m, 4H); 3.68 (bm, 2H); 3.30 (m, 3H); 2.65-2.41 (m, 6H); 2.31 (m, 2H); 1.87-1.19 (m, 73H); 0.90 (m, 12H).

AE. Compound 66: Heptadecan-9-yl 8-((6-((((3-hexylnonyl)oxy)carbonyl)oxy)hexyl)(2-hydroxyethyl)amino)octanoate

UPLC/ELSD: RT=3.16 min. MS ( _ES ) for _C49H97NO6 : m/z (MH ⁺ ) _797.683

¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.89 (p, 1H); 4.14 (m, 4H); 3.54 (bm, 2H); 2.68-2.38 (m, 6H); 2.30 (m, 2H); 1.77 -1.17 (m, 70H); 0.90 (m, 12H).

AF. Compound 67: 8-((3-((2-(methylamino)-3,4-dioxocyclobut-1-en-1-yl)amino)propyl)(6-((( (3-Pentyloctyl)oxy)carbonyl)oxy)hexyl)amino)heptadecan-9-yl octanoate

Compound 67 was prepared analogously to compound 65 using 6-undecanone in place of 7-tridecone. UPLC/ELSD: RT=3.04 min. MS (ES) for _C53H99N3O7 : _m /z (MH&lt _; ⁺ &gt;) _891.552 . ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 4.87 (p, 1H); 4.17 (m, 4H); 3.67 (bm, 2H); 3.28 (m, 3H); 2.68-2.38 (m, 6H); 2.31 (m, 2H); 1.86-1.18 (m, 69H); 0.90 (m, 12H).

Example 2: Sample formulation induced hEPO expression in mice and residual lipid levels in liver

To evaluate the expression potency and metabolic stability of the disclosed lipids, hepatocyte protein expression (hEPO) was measured following administration of the disclosed nanoparticles (eg, LNP-loaded) to mice.

Lipid nanoparticles (LNP) containing DSPC as phospholipid, cholesterol as structural lipid, PEG-1 as PEG lipid according to formula (A), (A1) were administered intravenously to CD-1 mice , (A2), (A3), (B), (I), (I'), (I-X), (I-Y), (I-Ya) (IA), (Ia), (Ib), (Ic) , (Id), (Ie), (If), (Ig), (II), (IIa) or (IIb), and mRNA encoding hEPO. The concentration of hEPO in serum was tested 6 hours after injection. The particles tested had a PDI between about 0.1-0.4, an encapsulation efficiency of about 82-99%, and a particle size of about 56-145 nm. All tested LNPs showed efficient delivery of mRNA to hepatocytes, with varying amounts of lipid remaining in mouse livers after 24 hours.

Table 2. hEPO expression and residual lipid levels in liver induced in mice by administration of LNPs comprising lipids of the present disclosure.

^aCalculation of the percentage of total dose assumes a 25g mouse with 1.5g liver

^b >95% equals very slow lipid metabolism

Enumerated Embodiments

Embodiment 1. A compound of formula (I1):

或其N-氧化物，

or its N-oxide,

or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中A是C_6-10芳基或杂环；并且R ⁴ is selected from free hydrogen, C _3-6 carbocyclic ring, -(CH ₂ ) _n Q, -(CH ₂ ) _n CHQR, -(CH ₂ ) _o C(R ¹² ) ₂ (CH ₂ ) _no Q, -CHQR , -CQ(R) ₂ , -C(O)NQR and unsubstituted C _1-6 alkyl groups, wherein Q is selected from carbocycle, heterocycle, -OR, -O(CH ₂ ) _n N( R) ₂ , -C(O)OR, -OC(O)R, -CX ₃ , -CX ₂ H, -CXH ₂ , -CN, -N(R) ₂ , -C(O)N(R) ₂ , -N(R)C(O)R, -N(R)S(O) ₂ R, -N(R)C(O)N(R) ₂ , -N(R)C(S)N (R) ₂ , -N(R)R ⁸ , -N(R)S(O) ₂ R ⁸ , -O(CH ₂ ) _n OR, -N(R)C(=NR ⁹ )N(R) ₂ , -N(R)C(=CHR ⁹ )N(R) ₂ , -OC(O)N(R) ₂ , -N(R)C(O)OR, -N(OR)C(O) R, -N(OR)S(O) ₂ R, -N(OR)C(O)OR, -N(OR)C(O)N(R) ₂ , -N(OR)C(S)N (R) ₂ , -N(OR)C(=NR ⁹ )N(R) ₂ , -N(OR)C(=CHR ⁹ )N(R) ₂ , -C(=NR ⁹ )N(R) ₂ , -C(=NR ⁹ )R, -C(O)N(R)OR, -(CH ₂ ) _n N(R) ₂ , -C(R)N(R) ₂ C(O)OR, NC(R)=R ¹¹ , N(C=NR ¹⁵ )R ¹¹ , NRC(C(O)NR ¹⁴ R ¹⁴ ′) ₂ , -NRC(O)(CH ₂ ) _p C(O)NR ¹⁴ R ¹⁴ 'and

wherein A is C _6-10 aryl or heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1, 2, 3 , 4 and 5; or

其中 ^R4 is

in

X ^a and X ^b are each independently O or S;

R ¹⁰ is selected from H, halo, -OH, R, -N(R) ₂ , -CN, -N ₃ , -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O) ₂ OR, -NO ₂ , -S(O) ₂ N(R) ₂ , -N(R)S (O) ₂ R, –NH(CH ₂ ) _t1 N(R) ₂ , –NH(CH ₂ ) _p1 O(CH ₂ ) _q1 N(R) ₂ , –NH(CH ₂ ) _s1 OR, –N( (CH ₂ ) _s1 OR) ₂ , -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R )(CH ₂ ) _{t1 -carbocycle} , -N(R)(CH ₂ ) _t1 -heterocycle, -N(R)(CH ₂ ) _t1 -aryl, -N(R)(CH ₂ ) _t1 -heterocycle the group consisting of aryl, carbocycle, heterocycle, aryl and heteroaryl;

n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

r is 0 or 1;

t ¹ is selected from 1, 2, 3, 4 and 5;

p ¹ is selected from 1, 2, 3, 4 and 5;

q ¹ is selected from 1, 2, 3, 4 and 5;

s ¹ is selected from 1, 2, 3, 4 and 5;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M is selected from -OC(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -OC(O)N(R ^M )-, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, SO-, -OS-, -S(R ^M ) ₂ O-, -OS( R ^M ) ₂ -, -S(O)O-, -OS(O)-, wherein M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) _2- , -S(R**) _2- or -S(O)-, wherein z is 1, 2, 3 or 4;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

R ⁸ is selected from the group consisting of C _3-6 carbocycles and heterocycles;

R ⁹ is selected from H, CN, NO ₂ , C _1-6 aryl, -OR, -S(O) ₂ R, -S(O) ₂ N(R) ₂ , C _2-6 alkenyl, C ₃ - the group consisting of ₆ carbocycles and heterocycles;

R ¹¹ is selected from the group consisting of C _3-6 carbocycles and heterocycles, wherein each of the C _3-6 carbocycles and heterocycles is optionally substituted with one or more R ¹³ ;

R ¹² is selected from the group consisting of H, OH, C _1-3 alkyl and C _2-3 alkenyl;

Each R ¹³ is independently selected from OH, oxo, halo, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _1-6 alkylamino, di- (C _1-6 alkyl) the group consisting of amino, amino, amido, cyano and nitro;

each R ¹⁴ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R ¹⁴ ' is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

R ¹⁵ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R is independently selected from the group consisting of _C1-6 alkyl, _C1-3 alkyl-aryl, _C2-3 alkenyl, and H;

R ^N is H or C _1-3 alkyl;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

Embodiment 2. A compound of formula (I'1):

或其N-氧化物，

or its N-oxide,

or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中A是C_6-10芳基或杂环；并且R ⁴ is selected from free hydrogen, C _3-6 carbocyclic ring, -(CH ₂ ) _n Q, -(CH ₂ ) _n CHQR, -(CH ₂ ) _o C(R ¹² ) ₂ (CH ₂ ) _no Q, -CHQR , -CQ(R) ₂ , -C(O)NQR and unsubstituted C _1-6 alkyl groups, wherein Q is selected from carbocycle, heterocycle, -OR, -O(CH ₂ ) _n N( R) ₂ , -C(O)OR, -OC(O)R, -CX ₃ , -CX ₂ H, -CXH ₂ , -CN, -N(R) ₂ , -C(O)N(R) ₂ , -N(R)C(O)R, -N(R)S(O) ₂ R, -N(R)C(O)N(R) ₂ , -N(R)C(S)N (R) ₂ , -N(R)R ⁸ , -N(R)S(O) ₂ R ⁸ , -O(CH ₂ ) _n OR, -N(R)C(=NR ⁹ )N(R) ₂ , -N(R)C(=CHR ⁹ )N(R) ₂ , -OC(O)N(R) ₂ , -N(R)C(O)OR, -N(OR)C(O) R, -N(OR)S(O) ₂ R, -N(OR)C(O)OR, -N(OR)C(O)N(R) ₂ , -N(OR)C(S)N (R) ₂ , -N(OR)C(=NR ⁹ )N(R) ₂ , -N(OR)C(=CHR ⁹ )N(R) ₂ , -C(=NR ⁹ )N(R) ₂ , -C(=NR ⁹ )R, -C(O)N(R)OR, -(CH ₂ ) _n N(R) ₂ , -C(R)N(R) ₂ C(O)OR, NC(R)=R ¹¹ , N(C=NR ¹⁵ )R ¹¹ , NRC(C(O)NR ¹⁴ R ¹⁴ ′) ₂ , -NRC(O)(CH ₂ ) _p C(O)NR ¹⁴ R ¹⁴ 'and

wherein A is C _6-10 aryl or heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1, 2, 3 , 4 and 5; or

其中 ^R4 is

in

X ^a and X ^b are each independently O or S;

R ¹⁰ is selected from H, halo, -OH, R, -N(R) ₂ , -CN, -N ₃ , -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O) ₂ OR, -NO ₂ , -S(O) ₂ N(R) ₂ , -N(R)S (O) ₂ R, –NH(CH ₂ ) _t1 N(R) ₂ , –NH(CH ₂ ) _p1 O(CH ₂ ) _q1 N(R) ₂ , –NH(CH ₂ ) _s1 OR, –N( (CH ₂ ) _s1 OR) ₂ , -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R )(CH ₂ ) _{t1 -carbocycle} , -N(R)(CH ₂ ) _t1 -heterocycle, -N(R)(CH ₂ ) _t1 -aryl, -N(R)(CH ₂ ) _t1 -heterocycle the group consisting of aryl, carbocycle, heterocycle, aryl and heteroaryl;

n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

r is 0 or 1;

t ¹ is selected from 1, 2, 3, 4 and 5;

p ¹ is selected from 1, 2, 3, 4 and 5;

q ¹ is selected from 1, 2, 3, 4 and 5;

s ¹ is selected from 1, 2, 3, 4 and 5;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M )- , -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, - C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, - OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, - (CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R** ) _2- or -S(O)-, wherein z is 1, 2, 3, or 4;

M' is selected from -OC(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -OC(O)N(R ^M )-, -N( R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, SO-, -OS-, -S(R ^M ) ₂ O-, -OS (R ^M ) ₂ -, -S(O)O-, -OS(O)-, wherein M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _{2- 13Alkenyl} , -B(R**)-, -Si(R**) _2- , -S(R**) _2- or -S(O)-, wherein z is 1, 2, 3, or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

R ⁸ is selected from the group consisting of C _3-6 carbocycles and heterocycles;

R ⁹ is selected from H, CN, NO ₂ , C _1-6 aryl, -OR, -S(O) ₂ R, -S(O) ₂ N(R) ₂ , C _2-6 alkenyl, C ₃ - the group consisting of ₆ carbocycles and heterocycles;

R ¹¹ is selected from the group consisting of C _3-6 carbocycles and heterocycles, wherein each of the C _3-6 carbocycles and heterocycles is optionally substituted with one or more R ¹³ ;

R ¹² is selected from the group consisting of H, OH, C _1-3 alkyl and C _2-3 alkenyl;

Each R ¹³ is independently selected from OH, oxo, halo, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _1-6 alkylamino, di- (C _1-6 alkyl) the group consisting of amino, amino, amido, cyano and nitro;

each R ¹⁴ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R ¹⁴ ' is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

R ¹⁵ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R is independently selected from the group consisting of _C1-6 alkyl, _C1-3 alkyl-aryl, _C2-3 alkenyl, and H;

R ^N is H or C _1-3 alkyl;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

Embodiment 3. A compound of formula (IX1):

或其N-氧化物，或它们的盐或异构体，其中：

or their N-oxides, or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中A是C_6-10芳基或杂环；并且R ⁴ is selected from free hydrogen, C _3-6 carbocyclic ring, -(CH ₂ ) _n Q, -(CH ₂ ) _n CHQR, -(CH ₂ ) _o C(R ¹² ) ₂ (CH ₂ ) _no Q, -CHQR , -CQ(R) ₂ , -C(O)NQR and unsubstituted C _1-6 alkyl groups, wherein Q is selected from carbocycle, heterocycle, -OR, -O(CH ₂ ) _n N( R) ₂ , -C(O)OR, -OC(O)R, -CX ₃ , -CX ₂ H, -CXH ₂ , -CN, -N(R) ₂ , -C(O)N(R) ₂ , -N(R)C(O)R, -N(R)S(O) ₂ R, -N(R)C(O)N(R) ₂ , -N(R)C(S)N (R) ₂ , -N(R)R ⁸ , -N(R)S(O) ₂ R ⁸ , -O(CH ₂ ) _n OR, -N(R)C(=NR ⁹ )N(R) ₂ , -N(R)C(=CHR ⁹ )N(R) ₂ , -OC(O)N(R) ₂ , -N(R)C(O)OR, -N(OR)C(O) R, -N(OR)S(O) ₂ R, -N(OR)C(O)OR, -N(OR)C(O)N(R) ₂ , -N(OR)C(S)N (R) ₂ , -N(OR)C(=NR ⁹ )N(R) ₂ , -N(OR)C(=CHR ⁹ )N(R) ₂ , -C(=NR ⁹ )N(R) ₂ , -C(=NR ⁹ )R, -C(O)N(R)OR, -(CH ₂ ) _n N(R) ₂ , -C(R)N(R) ₂ C(O)OR, NC(R)=R ¹¹ , N(C=NR ¹⁵ )R ¹¹ , NRC(C(O)NR ¹⁴ R ¹⁴ ′) ₂ , -NRC(O)(CH ₂ ) _p C(O)NR ¹⁴ R ¹⁴ 'and

wherein A is C _6-10 aryl or heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1, 2, 3 , 4 and 5; or

其中 ^R4 is

in

X ^a and X ^b are each independently O or S;

R ¹⁰ is selected from H, halo, -OH, R, -N(R) ₂ , -CN, -N ₃ , -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O) ₂ OR, -NO ₂ , -S(O) ₂ N(R) ₂ , -N(R)S (O) ₂ R, –NH(CH ₂ ) _t1 N(R) ₂ , –NH(CH ₂ ) _p1 O(CH ₂ ) _q1 N(R) ₂ , –NH(CH ₂ ) _s1 OR, –N( (CH ₂ ) _s1 OR) ₂ , -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R )(CH ₂ ) _{t1 -carbocycle} , -N(R)(CH ₂ ) _t1 -heterocycle, -N(R)(CH ₂ ) _t1 -aryl, -N(R)(CH ₂ ) _t1 -heterocycle the group consisting of aryl, carbocycle, heterocycle, aryl and heteroaryl;

n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

r is 0 or 1;

t ¹ is selected from 1, 2, 3, 4 and 5;

p ¹ is selected from 1, 2, 3, 4 and 5;

q ¹ is selected from 1, 2, 3, 4 and 5;

s ¹ is selected from 1, 2, 3, 4 and 5;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

R ⁸ is selected from the group consisting of C _3-6 carbocycles and heterocycles;

R ⁹ is selected from H, CN, NO ₂ , C _1-6 aryl, -OR, -S(O) ₂ R, -S(O) ₂ N(R) ₂ , C _2-6 alkenyl, C ₃ - the group consisting of ₆ carbocycles and heterocycles;

R ¹¹ is selected from the group consisting of C _3-6 carbocycles and heterocycles, wherein each of the C _3-6 carbocycles and heterocycles is optionally substituted with one or more R ¹³ ;

R ¹² is selected from the group consisting of H, OH, C _1-3 alkyl and C _2-3 alkenyl;

Each R ¹³ is independently selected from OH, oxo, halo, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _1-6 alkylamino, di- (C _1-6 alkyl) the group consisting of amino, amino, amido, cyano and nitro;

each R ¹⁴ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R ¹⁴ ' is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

R ¹⁵ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R is independently selected from the group consisting of _C1-6 alkyl, _C1-3 alkyl-aryl, _C2-3 alkenyl, and H;

R ^N is H or C _1-3 alkyl;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

Embodiment 4. The compound of any of the preceding embodiments, wherein at least one of M and M' is -OC(O)O-.

Embodiment 5. The compound of any one of the preceding embodiments, wherein at least one of M and M' is -OC(O)N(R ^M )- or -N(R ^M )C(O)O -.

Embodiment 6. The compound of any of the preceding embodiments, wherein at least one of M and ^M ' is -NRMC(O) ^NRM- .

Embodiment 7. The compound of any of the preceding embodiments, wherein at least one of M and M' is -ON=C(R ^M )-.

Embodiment 8. The compound of any one of the preceding embodiments, wherein at least one of M and M' is -OM"-O-, wherein each M" is independently selected from C _1-13 alkyl, C _2-13 alkenyl, -B(R**)- and -Si(R**) ₂ -.

Embodiment 9. The compound of any one of the preceding embodiments, wherein at least one of M and M' is -OM"-O-, and M" is _C1-3 alkyl.

Embodiment 10. The compound of any of the preceding embodiments, wherein M" is methyl.

Embodiment 11. The compound of any of the preceding embodiments, wherein M" is branched _C1-3 alkyl.

Embodiment 12. The compound of any of the preceding embodiments, wherein at least one of M and M' is -OM"-O-, and M" is -( _CH2 ) _zC (O)-.

Embodiment 13. The compound of any of the preceding embodiments, wherein z is 3.

Embodiment 14. The compound of any one of the preceding embodiments, wherein at least one of M and M' is

Embodiment 15. The compound of any one of the preceding embodiments, wherein at least one of M and M' is

Embodiment 16. The compound of any one of the preceding embodiments, wherein at least one of M and M' is -OM"-O-, wherein each M" is independently selected from -B(R**) - and -Si(R**) ₂ -.

Embodiment 17. The compound of any of the preceding embodiments, wherein each R** is _C1-3 alkyl.

Embodiment 18. The compound of any of the preceding embodiments, wherein each R** is methyl.

Embodiment 19. The compound of any of the preceding embodiments, wherein one of M and ^M ' is -NRMC(O) ^NRM- .

Embodiment 20. The compound of any of the preceding embodiments, wherein each R ^M is H.

Embodiment 21. The compound of any of the preceding embodiments, wherein R1 is R"^M'R' and R' is branched alkyl.

Embodiment 22. The compound of any of the preceding embodiments, wherein R1 is R"^M'R' and R' is alkenyl.

Embodiment 23. The compound of any of the preceding embodiments, wherein R1 is R"^M'R' and R" is branched alkyl.

Embodiment 24. The compound of any of the preceding embodiments, wherein R1 is R"^M'R' and R" is alkyl substituted with OH.

Embodiment 25. The compound of any of the preceding embodiments, wherein R' is R*YR*" and Y is cyclopropyl, cyclopentyl, or cyclohexyl.

Embodiment 26. The compound of any of the preceding embodiments, wherein R' is R*YR*" and R*" is _C2 alkyl, C3 _- alkyl, or _C4 alkyl.

Embodiment 27. The compound of any one of the preceding embodiments, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 28. The compound of any one of the preceding embodiments, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 29. The compound of one of the preceding claims, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 30. The compound of one of the preceding claims, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 31. The compound of one of the preceding claims, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 32. The compound of any one of the preceding Embodiments 1 or 2, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 33. The compound of any one of the preceding embodiments, wherein the compound has the structure:

其中R”是任选地被OH取代的直链或支链C₃-C₁₃烷基，并且m选自5、6、7、8和9。

wherein R" is a straight or branched C3 _{- C13} alkyl optionally substituted with OH, and m is selected from 5, 6, 7, 8 and 9.

Embodiment 34. A compound of formula (I-Y1):

或其N-氧化物，或它们的盐或异构体，其中：

or their N-oxides, or their salts or isomers, wherein:

R ¹ is selected from the group consisting of C _5-30 alkyl, C _5-20 alkenyl, -R*YR*", -YR*" and -R"M'R';

R ² and R ³ are independently selected from the group consisting of H, C _1-14 alkyl, C _2-14 alkenyl, -R*YR*", -YR*" and -R*OR*", or R ² and ^R3 together with the atoms to which it is attached form a heterocyclic or carbocyclic ring;

其中A是C_6-10芳基或杂环；并且R ⁴ is selected from free hydrogen, C _3-6 carbocyclic ring, -(CH ₂ ) _n Q, -(CH ₂ ) _n CHQR, -(CH ₂ ) _o C(R ¹² ) ₂ (CH ₂ ) _no Q, -CHQR , -CQ(R) ₂ , -C(O)NQR and unsubstituted C _1-6 alkyl groups, wherein Q is selected from carbocycle, heterocycle, -OR, -O(CH ₂ ) _n N( R) ₂ , -C(O)OR, -OC(O)R, -CX ₃ , -CX ₂ H, -CXH ₂ , -CN, -N(R) ₂ , -C(O)N(R) ₂ , -N(R)C(O)R, -N(R)S(O) ₂ R, -N(R)C(O)N(R) ₂ , -N(R)C(S)N (R) ₂ , -N(R)R ⁸ , -N(R)S(O) ₂ R ⁸ , -O(CH ₂ ) _n OR, -N(R)C(=NR ⁹ )N(R) ₂ , -N(R)C(=CHR ⁹ )N(R) ₂ , -OC(O)N(R) ₂ , -N(R)C(O)OR, -N(OR)C(O) R, -N(OR)S(O) ₂ R, -N(OR)C(O)OR, -N(OR)C(O)N(R) ₂ , -N(OR)C(S)N (R) ₂ , -N(OR)C(=NR ⁹ )N(R) ₂ , -N(OR)C(=CHR ⁹ )N(R) ₂ , -C(=NR ⁹ )N(R) ₂ , -C(=NR ⁹ )R, -C(O)N(R)OR, -(CH ₂ ) _n N(R) ₂ , -C(R)N(R) ₂ C(O)OR, NC(R)=R ¹¹ , N(C=NR ¹⁵ )R ¹¹ , NRC(C(O)NR ¹⁴ R ¹⁴ ′) ₂ , -NRC(O)(CH ₂ ) _p C(O)NR ¹⁴ R ¹⁴ 'and

wherein A is C _6-10 aryl or heterocycle; and

each o is independently selected from 1, 2, 3, and 4; p is 0, 1, 2, 3, or 4; q is 1, 2, 3, or 4; and each n is independently selected from 1, 2, 3 , 4 and 5; or

其中 ^R4 is

in

X ^a and X ^b are each independently O or S;

R ¹⁰ is selected from H, halo, -OH, R, -N(R) ₂ , -CN, -N ₃ , -C(O)OH, -C(O)OR, -OC(O)R, -OR, -SR, -S(O)R, -S(O)OR, -S(O) ₂ OR, -NO ₂ , -S(O) ₂ N(R) ₂ , -N(R)S (O) ₂ R, –NH(CH ₂ ) _t1 N(R) ₂ , –NH(CH ₂ ) _p1 O(CH ₂ ) _q1 N(R) ₂ , –NH(CH ₂ ) _s1 OR, –N( (CH ₂ ) _s1 OR) ₂ , -N(R)-carbocycle, -N(R)-heterocycle, -N(R)-aryl, -N(R)-heteroaryl, -N(R )(CH ₂ ) _{t1 -carbocycle} , -N(R)(CH ₂ ) _t1 -heterocycle, -N(R)(CH ₂ ) _t1 -aryl, -N(R)(CH ₂ ) _t1 -heterocycle the group consisting of aryl, carbocycle, heterocycle, aryl and heteroaryl;

n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

r is 0 or 1;

t ¹ is selected from 1, 2, 3, 4 and 5;

p ¹ is selected from 1, 2, 3, 4 and 5;

q ¹ is selected from 1, 2, 3, 4 and 5;

s ¹ is selected from 1, 2, 3, 4 and 5;

each R ⁵ is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H;

each R is independently selected from the group consisting of OH, C _1-3 alkyl, C _2-3 alkenyl and H ^;

M' is selected from -OC(O)O-, -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -OC(O)-NR ^M -C(O)O-, -OM"-O-, -C(O)N(R ^M )-, -N(R ^M )C(O)-, -OC(O)N(R ^M ) -, -N(R ^M )C(O)O-, -NR ^M C(O)NR ^M -, -ON=C(R ^M )-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR ^M )O-, -S(O) ₂ -, -SS-, -SO-, -OS-, S(R ^M ) ₂ O-, -OS(R ^M ) ₂ -, -S(O)O-, -OS(O)-, aryl and heteroaryl, where M" is a bond, -(CH ₂ ) _z C(O)-, C _1-13 alkyl, C _2-13 alkenyl, -B(R**)-, -Si(R**) ₂ -, -S(R* *) _2- or -S(O)-, where z is 1, 2, 3 or 4;

R ⁷ is selected from the group consisting of C _1-3 alkyl, C _2-3 alkenyl and H;

R ⁸ is selected from the group consisting of C _3-6 carbocycles and heterocycles;

R ⁹ is selected from H, CN, NO ₂ , C _1-6 aryl, -OR, -S(O) ₂ R, -S(O) ₂ N(R) ₂ , C _2-6 alkenyl, C ₃ - the group consisting of ₆ carbocycles and heterocycles;

R ¹¹ is selected from the group consisting of C _3-6 carbocycles and heterocycles, wherein each of the C _3-6 carbocycles and heterocycles is optionally substituted with one or more R ¹³ ;

R ¹² is selected from the group consisting of H, OH, C _1-3 alkyl and C _2-3 alkenyl;

Each R ¹³ is independently selected from OH, oxo, halo, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyl, C _1-6 alkylamino, di- (C _1-6 alkyl) the group consisting of amino, amino, amido, cyano and nitro;

each R ¹⁴ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R ¹⁴ ' is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

R ¹⁵ is independently selected from the group consisting of H, OH, C _1-6 alkyl and C _2-3 alkenyl;

each R is independently selected from the group consisting of _C1-6 alkyl, _C1-3 alkyl-aryl, _C2-3 alkenyl, and H;

R ^N is H or C _1-3 alkyl;

each R' is independently selected from the group consisting of C _1-18 alkyl, C _2-18 alkenyl, -R*YR*", -YR*", (CH ₂ ) _q OR* and H,

and each q is independently selected from 1, 2 and 3;

each R ^M is independently selected from the group consisting of H, C _1-6 alkyl and C _2-6 alkenyl;

each R" is independently selected from the group consisting of C _3-15 alkyl and C _3-15 alkenyl;

each R*" is independently selected from the group consisting of C _1-15 alkyl and C _2-15 alkenyl;

each R* is independently selected from the group consisting of C _1-12 alkyl and C _2-12 alkenyl;

each R** is independently selected from the group consisting of H, OH, C _1-12 alkyl, C _2-12 alkenyl, (CH ₂ ) _q OR*, and (CH ₂ ) _q OH;

each Y is independently a _C3-6 carbocycle;

each X is independently selected from the group consisting of F, Cl, Br, and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13.

其中每个R”独立地选自H、OH和C_1-6烷基，l选自2、3、4、5和6，并且m选自5、6、7、8和9。Embodiment 35. The compound of any one of the preceding embodiments, wherein the compound has the structure:

wherein each R" is independently selected from H, OH, and _C1-6 alkyl, l is selected from 2, 3, 4, 5, and 6, and m is selected from 5, 6, 7, 8, and 9.

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；Embodiment 36. The compound of any one of the preceding embodiments, wherein R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ , and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

Embodiment 37. A compound of formula (A):

或其N-氧化物，或它们的盐或异构体，其中

or their N-oxides, or their salts or isomers, wherein

m is selected from 5, 6, 7, 8 and 9;

R ² and R ³ are each independently selected from the group consisting of H, C _1-14 alkyl and C _2-14 alkenyl;

其中n2选自1、2、3、4、5、6、7、8、9和10；并且R¹⁰是-N(R)₂，其中每个R独立地选自由C_1-6烷基、C_2-3烯基和H组成的组；R4 is selected from -( _CH2 )nOH, wherein _n is selected from 1, 2, 3, ⁴ and 5; and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and ¹⁰ ; and R10 is -N(R) ₂ , wherein each R is independently selected from _C1-6 alkyl, The group consisting of C _2-3 alkenyl and H;

M and M' are each independently selected from -OC(O)O- and -C(O)O-;

wherein at least one of M and M' is -OC(O)O-;

R' ^a is C _1-18 alkyl or C _2-18 alkenyl; and

R" is C3 _{- C13} alkyl optionally substituted with OH.

其中

表示与N的连接点，并且

表示与M的连接点。Embodiment 38. The compound of any one of the preceding embodiments, wherein R" is selected from

in

represents the junction with N, and

Indicates the connection point with M.

Embodiment 39. The compound of any one of the preceding embodiments, wherein the compound has one of the following structures:

其中

表示连接点；Embodiment 40. The compound of any one of the preceding embodiments, wherein ^R'a is:

in

represents a connection point;

R ^aα , R ^aβ and R ^aγ are each independently selected from the group consisting of H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^aα , R ^aβ and R ^aγ is selected from C _1- the group consisting of ₁₂ alkyl and C _2-12 alkenyl; and

R' is C _1-12 alkyl or C _2-12 alkenyl.

Embodiment 41. The compound of any one of the preceding embodiments, wherein R ^aβ and R ^{aγ are each H and R aα is} C _1-6 alkyl.

Embodiment 42. The compound of any one of the preceding embodiments, wherein R ^aα and R ^aγ are each H and R ^aβ is C _1-6 alkyl.

Embodiment 43. The compound of any one of the preceding embodiments, wherein R ^aβ is C ₂ alkyl.

Embodiment 44. The compound of any one of the preceding embodiments, wherein R ^aα and R ^aβ are each H and R ^aγ is C ^1-6 _alkyl .

Embodiment 45. The compound of any one of the preceding embodiments, wherein R ^aγ is C ₅ or C ₆ alkyl.

Embodiment 46. The compound of any of the preceding embodiments, wherein R' is _C5 or _C6 alkyl.

Embodiment 47. The compound of any one of the preceding embodiments, wherein the compound has the structure:

Embodiment 48. The compound of any of the preceding embodiments, wherein R4 is ^- ( _CH2 ) _nOH .

Embodiment 49. The compound of any of the preceding embodiments, wherein R4 is ^- ( _{CH2 )} 2OH.

Embodiment 50. The compound of any one of the preceding embodiments, wherein R ⁴ is

Embodiment 51. The compound of any one of the preceding embodiments, wherein R ⁴ is

Embodiment 52. The compound of any of the preceding embodiments, wherein n2 is 2 or 4.

Embodiment 53. A compound selected from:

Embodiment 54. An empty lipid nanoparticle (empty LNP) comprising the compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid.

Embodiment 55. A lipid-loaded nanoparticle (loaded LNP) comprising a compound of any of the preceding embodiments, a phospholipid, a structured lipid, a PEG lipid, and one or more therapeutic agents and / or prophylactics.

Embodiment 56. The empty or loaded LNP of any one of the preceding embodiments, comprising the compound in an amount from about 40% to about 60%.

Embodiment 57. The empty or loaded LNP of any one of the preceding embodiments, comprising phospholipid in an amount from about 0% to about 20%.

Embodiment 58. The empty or loaded LNP of any one of the preceding embodiments, comprising structured lipids in an amount from about 30% to about 50%.

Embodiment 59. The empty LNP or loaded LNP of any of the preceding embodiments, comprising PEG lipids in an amount from about 0% to about 5%.

Embodiment 60. The empty or supported LNP of any of the preceding embodiments, comprising from about 40 mol % to about 60 mol % of the compound of any preceding embodiment, from about 0 mol % to about 20 mol % % phospholipids, about 30 mol % to about 50 mol % structured lipids, and about 0 mol % to about 5 mol % PEG lipids.

Embodiment 61. The empty LNP or supported LNP of any of the preceding embodiments, comprising from about 30 mol % to about 60 mol % of the compound of any preceding embodiment, from about 0 mol % to about 30 mol % % phospholipids, about 18.5 mol % to about 48.5 mol % structured lipids, and about 0 mol % to about 10 mol % PEG lipids.

Embodiment 62. The loaded LNP of any of the preceding embodiments, wherein the one or more therapeutic and/or prophylactic agents are polynucleotides or polypeptides.

Embodiment 63. The loaded LNP of any of the preceding embodiments, wherein the one or more therapeutic and/or prophylactic agents is a nucleic acid.

Embodiment 64. The loaded LNP of any of the preceding embodiments, wherein the one or more therapeutic and/or prophylactic agents are selected from the group consisting of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

Embodiment 65. The loaded LNP of any one of the preceding embodiments, wherein the DNA is selected from the group consisting of double-stranded DNA, single-stranded DNA (ssDNA), partially double-stranded DNA, triple-stranded DNA, and partially triple-stranded DNA Group.

Embodiment 66. The loaded LNP of any of the preceding embodiments, wherein the DNA is selected from the group consisting of circular DNA, linear DNA, and mixtures thereof.

Embodiment 67. The loaded LNP of any one of the preceding embodiments, wherein the one or more therapeutic and/or prophylactic agents are selected from the group consisting of plasmid expression vectors, viral expression vectors, and mixtures thereof.

Embodiment 68. The loaded LNP of any of the preceding embodiments, wherein the one or more therapeutic and/or prophylactic agents is RNA.

Embodiment 69. The loaded LNP of any of the preceding embodiments, wherein the RNA is selected from the group consisting of single-stranded RNA, double-stranded RNA (dsRNA), partially double-stranded RNA, and mixtures thereof.

Embodiment 70. The loaded LNP of any of the preceding embodiments, wherein the RNA is selected from the group consisting of circular RNA, linear RNA, and mixtures thereof.

Embodiment 71. The loaded LNP of any of the preceding embodiments, wherein the RNA is selected from the group consisting of short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecule , microRNA (miRNA), antagomir, antisense RNA, ribozyme, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

Embodiment 72. The loaded LNP of any of the preceding embodiments, wherein the RNA is mRNA.

Embodiment 73. The loaded LNP of any of the preceding embodiments, wherein the mRNA is a modified mRNA (mmRNA).

Embodiment 74. The loaded LNP of any of the preceding embodiments, wherein the mRNA incorporates a microRNA binding site (miR binding site).

Embodiment 75. The load LNP of any one of the preceding embodiments, wherein the mRNA comprises one of a stem loop, a chain termination nucleoside, a polyA sequence, a polyadenylation signal and/or a 5' cap structure or multiple.

Embodiment 76. The empty LNP or loaded LNP of any one of the preceding embodiments, wherein the phospholipid is selected from the group consisting of:

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),

1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC),

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),

1,2-Di-undecanoyl-sn-glycero-phosphocholine (DUPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),

1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC),

1-Oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC),

1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

1,2-Dilinolenoyl-sn-glycero-3-phosphocholine,

1,2-Diarachidonoyl-sn-glycero-3-phosphocholine,

1,2-Di-docosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytate Alkanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE),

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine,

1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine,

1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine,

1,2-Di-docosahexaenoyl-sn-glycero-3-phosphoethanolamine,

1,2-Dioleoyl-sn-glycero-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

Embodiment 77. The empty or loaded LNP of any of the preceding embodiments, wherein the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

Embodiment 78. The empty or loaded LNP of any one of the preceding embodiments, wherein the structural lipid is selected from the group consisting of cholesterol, coprosterol, sitosterol, ergosterol, rapeseed oil sterols, stigmasterol, brassicasterol and mixtures thereof.

或其盐。Embodiment 79. The empty LNP or loaded LNP of any one of the preceding embodiments, wherein the structural lipid is

or its salt.

或其盐。Embodiment 80. The empty or loaded LNP of any one of the preceding embodiments, wherein the structural lipid is cholesterol:

or its salt.

Embodiment 81. The empty or loaded LNP of any of the preceding embodiments, wherein the PEG lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.

Embodiment 82. The empty LNP or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is selected from the group consisting of 1,2- dimyristoyl-sn-glyceromethoxypoly Ethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)](PEG-DSPE), PEG-distearyl Glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycinamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG- DPPE) or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

Embodiment 83. The empty or loaded LNP of any of the preceding embodiments, wherein the PEG lipid is PEG-DMG.

Embodiment 84. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of formula (PL-I):

or a salt thereof, where:

R ^3PL1 is –OR ^OPL1 ;

R ^OPL1 is hydrogen, an optionally substituted alkyl or oxygen protecting group;

r ^PL1 is an integer between 1 and 100, inclusive;

L ¹ is an optionally substituted C _1-10 alkylene group, wherein at least one methylene group in the optionally substituted C _1-10 alkylene group is independently replaced by: optionally substituted carbocyclylene, any optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(R ^NPL1 ), S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), C(O)O, OC(O), OC(O)O, OC(O)N(R ^NPL1 ), NR ^NPL1 C(O)O or NR ^NPL1 C(O)N( R ^NPL1 );

D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions;

m ^PL1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A has the following formula:

Each instance of L is ^{independently} a bond or an optionally substituted _C1-6 alkylene where one methylene unit in the optionally substituted _C1-6 alkylene is optionally replaced with: O , N(R ^NPL1 ), S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), C(O)O, OC(O), OC(O)O, OC( O)N(R ^NPL1 ), NR ^NPL1 C(O)O or NR ^NPL1 C(O)N(R ^NPL1 );

Each instance of R ^2SL is independently optionally substituted C _1-30 alkyl, optionally substituted C _1-30 alkenyl, or optionally substituted C _1-30 alkynyl; optionally one of wherein R ^2SL or more methylene units are independently replaced with: optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N( R ^NPL1 ), O, S, C(O), C(O)N(R ^NPL1 ), NR ^NPL1 C(O), NR ^NPL1 C(O)N(R ^NPL1 ), C(O)O, OC( O), OC(O)O, OC(O)N(R ^NPL1 ), NR ^NPL1 C(O)O, C(O)S, SC(O), C(=NR ^NPL1 ), C(=NR ^NPL1 )N(R ^NPL1 ), NR ^NPL1 C(=NR ^NPL1 ), NR ^NPL1 C(=NR ^NPL1 )N(R ^NPL1 ), C(S), C(S)N(R ^NPL1 ), NR ^NPL1 C(S ), NR ^NPL1 C(S)N(R ^NPL1 ), S(O), OS(O), S(O)O, OS(O)O, OS(O) ₂ , S(O) ₂ O, OS (O) ₂ O, N(R ^NPL1 )S(O), S(O)N(R ^NPL1 ), N(R ^NPL1 )S(O)N(R ^NPL1 ), OS(O)N(R ^NPL1 ) , N(R ^NPL1 )S(O)O, S(O) ₂ , N(R ^NPL1 )S(O) ₂ , S(O) ₂ N(R ^NPL1 ), N(R ^NPL1 )S(O) ₂ N(R ^NPL1 ), OS(O) ₂ N(R ^NPL1 ) or N(R ^NPL1 )S(O) ₂ O;

Each instance of R ^NPL1 is independently a hydrogen, optionally substituted alkyl or nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

^pSL is 1 or 2.

或其盐。Embodiment 85. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of formula (PL-I-OH):

or its salt.

或其盐或异构体，其中：Embodiment 86. The empty LNP or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of formula (PL-II-OH):

or a salt or isomer thereof, wherein:

R ^3PEG is -OR ^O ;

R ^O is a hydrogen, C _1-6 alkyl or oxygen protecting group;

r ^PEG is an integer between 1 and 100;

R ^5PEG is C _10-40 alkyl, C _10-40 alkenyl, or optionally substituted C _10-40 alkynyl; and optionally one or more methylene groups of R ^5PEG are independently replaced with: C _{3 -10} -membered carbocyclylene, 4- to 10-membered heterocyclylene, C _6-10 -membered arylene, 4- to 10-membered heteroarylene, -N(R ^NPEG )-, -O-, -S-, - C(O)–, –C(O)N(R ^NPEG )–, –NR ^NPEG C(O)–, –NR ^NPEG C(O)N(R ^NPEG )–, –C(O)O–, – OC(O)–, –OC(O)O–, –OC(O)N(R ^NPEG )–, –NR ^NPEG C(O)O–, –C(O)S–, –SC(O)– , –C(=NR ^NPEG )–, –C(=NR ^NPEG )N(R ^NPEG )–, –NR ^NPEG C(=NR ^NPEG )–, –NR ^NPEG C(=NR ^NPEG )N(R ^NPEG )– , –C(S)–, –C(S)N(R ^NPEG )–, –NR ^NPEG C(S)–, –NR ^NPEG C(S)N(R ^NPEG )–, –S(O)–, –OS(O)–, –S(O)O–, –OS(O)O–, –OS(O) ₂ –, –S(O) ₂ O–, –OS(O) ₂ O–, – N(R ^NPEG )S(O)–, –S(O)N(R ^NPEG )–, –N(R ^NPEG )S(O)N(R ^NPEG )–, –OS(O)N(R ^NPEG ) –, –N(R ^NPEG )S(O)O–, –S(O) ₂ –, –N(R ^NPEG )S(O) ₂ –, –S(O) ₂ N(R ^NPEG )–, – N(R ^NPEG )S(O) ₂ N(R ^NPEG )–, –OS(O) ₂ N(R ^NPEG )– or –N(R ^NPEG )S(O) ₂ O–; and

Each instance of R ^NPEG is independently a hydrogen, _C1-6 alkyl or nitrogen protecting group.

Embodiment 87. The empty or supported LNP of any one of the preceding embodiments, wherein the PEG lipid of formula (PL-II-OH), r is an integer between 40 and 50.

Embodiment 88. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid of formula (PL-II-OH), r is 45.

Embodiment 89. The empty or supported LNP of any one of the preceding embodiments, wherein the PEG lipid of formula (PL-II ^- OH), R5 is _C17 alkyl.

其中r^PEG是介于1与100之间的整数。Embodiment 90. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid of formula (PL-II):

where ^rPEG is an integer between 1 and 100.

Embodiment 91. The empty LNP or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of formula (PEG-1):

或其盐或异构体，其中s^PL1是介于1与100之间的整数。Embodiment 92. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of formula (PL-III):

or a salt or isomer thereof, wherein s ^PL1 is an integer between 1 and 100.

Embodiment 93. The empty or loaded LNP of any one of the preceding embodiments, wherein the PEG lipid is a compound of the formula:

Embodiment 94. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the The structural lipid is cholesterol.

Embodiment 95. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the structured lipid is Cholesterol and the PEG lipid was PEG _2k -DMG.

Embodiment 96. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the structured lipid is cholesterol and the PEG lipid is PEG-1.

Embodiment 97. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the The PEG lipid is PEG _2k -DMG.

Embodiment 98. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC and the The PEG lipid is PEG-1.

Embodiment 99. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, wherein the The structural lipid is cholesterol and the PEG lipid is PEG _2k -DMG.

Embodiment 100. An empty lipid nanoparticle (empty LNP) comprising the compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, wherein the phospholipid is DSPC, wherein the The structural lipid is cholesterol and the PEG lipid is PEG-1.

Embodiment 101. A lipid-loaded nanoparticle (loaded LNP) comprising a compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid and one or more therapeutic agents and /or prophylactic, wherein the phospholipid is DSPC and the structural lipid is cholesterol.

Embodiment 102. A lipid-loaded nanoparticle (loaded LNP) comprising the compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, and one or more therapeutic agents and /or prophylactic, wherein the structural lipid is cholesterol and the PEG lipid is PEG _2k -DMG.

Embodiment 103. A lipid-loaded nanoparticle (loaded LNP) comprising a compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, and one or more therapeutic agents and /or prophylactic, wherein the structural lipid is cholesterol and the PEG lipid is PEG-1.

Embodiment 104. A lipid-loaded nanoparticle (loaded LNP) comprising the compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, and one or more therapeutic agents and /or prophylactic, wherein the phospholipid is DSPC and the PEG lipid is PEG _2k -DMG.

Embodiment 105. A lipid-loaded nanoparticle (loaded LNP) comprising the compound of any of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid, and one or more therapeutic agents and /or prophylactic, wherein the phospholipid is DSPC and the PEG lipid is PEG-1.

Embodiment 106. A lipid-loaded nanoparticle (loaded LNP) comprising a compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid and one or more therapeutic agents and /or prophylactic, wherein the phospholipid is DSPC, the structural lipid is cholesterol and the PEG lipid is PEG _2k -DMG.

Embodiment 107. A lipid-loaded nanoparticle (loaded LNP) comprising a compound of any one of the preceding embodiments, a phospholipid, a structured lipid, and a PEG lipid and one or more therapeutic agents and /or prophylactic, wherein the phospholipid is DSPC, the structural lipid is cholesterol and the PEG lipid is PEG-1.

Embodiment 108. The empty or loaded LNP of any one of the preceding embodiments, comprising DSPC in an amount from about 0% to about 20%.

Embodiment 109. The empty or loaded LNP of any of the preceding embodiments, comprising cholesterol in an amount from about 30% to about 50%.

Embodiment 110. The empty or loaded LNP of any of the preceding embodiments, comprising _PEG2k -DMG in an amount of about 0% to about 5%.

Embodiment 111. The empty or loaded LNP of any of the preceding embodiments, comprising PEG-1 in an amount from about 0% to about 5%.

Embodiment 112. The empty or supported LNP of any of the preceding embodiments, comprising from about 40 mol % to about 60 mol % of the compound of any preceding embodiment; from about 0 mol % to about 20 mol % % DSPC; about 30 mol % to about 50 mol % cholesterol; and about 0 mol % to about 5 mol % PEG _2k -DMG.

Embodiment 113. The empty LNP or loaded LNP of any preceding embodiment, comprising from about 40 mol % to about 60 mol % of the compound of any preceding embodiment; from about 0 mol % to about 20 mol % % DSPC; about 30 mol % to about 50 mol % cholesterol; and about 0 mol % to about 5 mol % PEG-1.

Embodiment 114. The loaded LNP of any one of the preceding embodiments, the encapsulation efficiency of the therapeutic and/or prophylactic agent is between 80% and 100%.

Embodiment 115. The loaded LNP of any of the preceding embodiments, wherein the weight/weight ratio of the lipid component to the mRNA is from about 10:1 to about 60:1.

Embodiment 116. The loaded LNP of any of the preceding embodiments, wherein the weight/weight ratio of the lipid component to the mRNA is 20:1.

Embodiment 117. The supported LNP of any of the preceding embodiments, wherein the N:P ratio is from about 5:1 to about 8:1.

Embodiment 118. A pharmaceutical composition comprising the loaded LNP of any of the preceding embodiments and a pharmaceutically acceptable carrier.

Embodiment 119. The pharmaceutical composition of any of the preceding embodiments, further comprising a cryoprotectant, a buffer, or a combination thereof.

Embodiment 120. The pharmaceutical composition of any of the preceding embodiments, wherein the cryoprotectant comprises sucrose.

Embodiment 121. The pharmaceutical composition of any of the preceding embodiments, wherein the cryoprotectant comprises sodium acetate.

Embodiment 122. The pharmaceutical composition of any of the preceding embodiments, wherein the cryoprotectant comprises sucrose and sodium acetate.

Embodiment 123. The pharmaceutical composition of any of the preceding embodiments, wherein the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, and tris buffer agent.

Embodiment 124. A method of delivering a therapeutic and/or prophylactic agent to cells in a subject, the method comprising administering to the subject the loaded LNP of any of the preceding embodiments.

Embodiment 125. A method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a subject, the method comprising administering to the subject the loaded LNP of any one of the preceding embodiments .

Embodiment 126. A method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue in a subject, the method comprising administering to the subject the method of any of the preceding embodiments Load LNP.

Embodiment 127. A method of producing a polypeptide of interest in a cell in a subject, the method comprising administering to the subject the loaded LNP of any of the preceding embodiments.

Embodiment 128. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the loaded LNP of any of the preceding embodiments.

Embodiment 129. Use of the loaded LNP of any of the preceding embodiments in the manufacture of a medicament for delivering a therapeutic and/or prophylactic agent to cells in a subject.

Embodiment 130. Use of the loaded LNP of any of the preceding embodiments in the manufacture of a medicament for the specific delivery of a therapeutic and/or prophylactic agent to an organ of a subject.

Embodiment 131. Use of the loaded LNP of any of the preceding embodiments in the manufacture of a medicament for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue of a subject.

Embodiment 132. Use of the loaded LNP of any of the preceding embodiments in the manufacture of a medicament for producing a polypeptide of interest in cells in a subject.

Embodiment 133. Use of the loaded LNP of any of the preceding embodiments in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.

Embodiment 134. The loaded LNP of any one of the preceding embodiments for use in delivering a therapeutic and/or prophylactic agent to a cell in a subject, wherein the delivery comprises administering to the subject A therapeutically effective amount of the loaded LNP.

Embodiment 135. The load LNP of any one of the preceding embodiments for use in the specific delivery of a therapeutic agent and/or a prophylactic agent to an organ of a subject, wherein the delivery comprises to the subject A therapeutically effective amount of the loaded LNP is administered.

Embodiment 136. The loaded LNP of any one of the preceding embodiments for enhanced delivery of a therapeutic and/or prophylactic agent to a subject's target tissue, wherein the use comprises delivering to the subject A loaded LNP according to any of the preceding embodiments is administered.

Embodiment 137. The load LNP of any one of the preceding embodiments, which is used to produce a polypeptide of interest in a cell in a subject, the use comprising administering to the subject the use of the aforementioned embodiments The load LNP of any one.

Embodiment 138. The load LNP of any one of the preceding embodiments for use in the treatment of a disease or condition in a subject in need, wherein the treatment comprises administering to the subject a therapeutically effective amount of the load LNP.

Embodiment 139. A method of delivering a therapeutic and/or prophylactic agent to cells in a subject, the method comprising administering to the subject the pharmaceutical composition of any of the preceding embodiments .

Embodiment 140. A method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a subject, the method comprising administering to the subject the pharmaceutical combination of any of the preceding embodiments thing.

Embodiment 141. A method for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue in a subject, the method comprising administering to the subject the method of any of the preceding embodiments pharmaceutical composition.

Embodiment 142. A method of producing a polypeptide of interest in a cell in a subject, the method comprising administering to the subject the loaded LNP of any of the preceding embodiments.

Embodiment 143. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any of the preceding embodiments.

Embodiment 144. Use of the pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for delivering a therapeutic and/or prophylactic agent to cells in a subject.

Embodiment 145. Use of the pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for the specific delivery of a therapeutic and/or prophylactic agent to an organ of a subject.

Embodiment 146. Use of the pharmaceutical composition of any of the preceding embodiments for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue in a subject, the method comprising administering to the subject The pharmaceutical composition of any of the preceding embodiments is administered.

Embodiment 147. Use of the pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for producing a polypeptide of interest in cells in a subject.

Embodiment 148. Use of the pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.

Embodiment 149. The pharmaceutical composition of any one of the preceding embodiments, for use in delivering a therapeutic and/or prophylactic agent to cells in a subject, wherein the delivery comprises delivering to the subject A therapeutically effective amount of the pharmaceutical composition is administered.

Embodiment 150. The pharmaceutical composition of any one of the preceding embodiments, for use in the specific delivery of a therapeutic and/or prophylactic agent to an organ of a subject, wherein the delivery comprises delivering to the subject is administered a therapeutically effective amount of the pharmaceutical composition.

Embodiment 151. The pharmaceutical composition of any one of the preceding embodiments for enhanced delivery of a therapeutic and/or prophylactic agent to a target tissue in a subject, wherein the using comprises delivering to the subject to administer the pharmaceutical composition of any of the preceding embodiments.

Embodiment 152. The pharmaceutical composition of any one of the preceding embodiments, which is used to produce a polypeptide of interest in a cell in a subject, the use comprising administering to the subject as described previously The pharmaceutical composition of any one of the schemes.

Embodiment 153. The pharmaceutical composition of any one of the preceding embodiments, for use in the treatment of a disease or disorder in a subject in need thereof, wherein the treatment comprises administering to the subject a therapeutically effective amount of the pharmaceutical composition.

Embodiment 154. The method, use or loaded LNP or pharmaceutical composition for use of any of the preceding embodiments, wherein the organ is selected from the group consisting of liver, kidney, spleen or lung.

Embodiment 155. The method, use or loaded LNP or pharmaceutical composition for use of any of the preceding embodiments, wherein the target tissue is selected from the group consisting of liver, kidney, spleen or lung.

Embodiment 156. The method, use or loaded LNP or pharmaceutical composition for use of any of the preceding embodiments, wherein the administering is parenterally.

Embodiment 157. The method or loaded LNP or pharmaceutical composition for use of any one of the preceding embodiments, wherein the administration is intramuscular, intradermal, subcutaneous and/or intravenous.

Embodiment 158. The use of any of the preceding embodiments, wherein the medicament is for parenteral administration.

Embodiment 159. The use of any of the preceding embodiments, wherein the medicament is for intramuscular, intradermal, subcutaneous and/or intravenous administration.

Embodiment 160. The method, use or loaded LNP or pharmaceutical composition for use of any of the preceding embodiments, wherein the subject is a human.

Equivalent scheme

It should be understood that, while the present disclosure has been described in conjunction with the detailed description of the present disclosure, the foregoing description is intended to illustrate, and not to limit, the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims (32)

1. A compound of formula (A):

or an N-oxide thereof, or a salt or isomer thereof, wherein

m is selected from 5, 6, 7, 8 and 9;

R²and R³Each independently selected from the group consisting of H, C_1-14Alkyl and C_2-14Alkenyl groups;

R₄is selected from- (CH)₂)_nOH, wherein n is selected from 1, 2, 3, 4 and 5, and

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and R is¹⁰is-N (R)₂Wherein each R is independently selected from the group consisting of C_1-6Alkyl radical, C_2-3Alkenyl and H;

m and M' are each independently selected from the group consisting of-OC (O) O-and-C (O) O-;

wherein at least one of M and M' is-OC (O) O-;

R’^ais C_1-18Alkyl or C_2-18An alkenyl group; and is

R' is C optionally substituted by OH₃-C₁₃An alkyl group.

2. The compound of claim 1, wherein R "is selected from

Wherein

Represents a point of attachment to N, and

indicating the point of attachment to M.

3. The compound of claim 1 or 2, wherein the compound has one of the following structures:

4. the compound of any preceding claim, wherein R'^aThe method comprises the following steps:

wherein

Represents a connection point;

R^aα、R^aβand R^aγEach independently selected from the group consisting of H, C_1-12Alkyl and C_2-12Alkenyl, wherein R^aα、R^aβAnd R^aγAt least one of them is selected from C_1-12Alkyl and C_2-12Alkenyl groups; and is

R' is C _1-12Alkyl or C_2-12An alkenyl group.

5. The compound of claim 4, wherein R^aβAnd R^aγEach is H and R^aαIs C_1-6An alkyl group.

6. The compound of claim 4, wherein R^aαAnd R^aγEach is H and R^aβIs C_1-6An alkyl group.

7. The compound of claim 6, wherein R^aβIs C₂An alkyl group.

8. The compound of claim 4, wherein R^aαAnd R^aβEach is H and R^aγIs C_1-6An alkyl group.

9. The compound of claim 8, wherein R^aγIs C₅Or C₆An alkyl group.

10. The compound of any one of the preceding claims, wherein R' is C₅Or C₆An alkyl group.

11. The compound of any one of the preceding claims, wherein the compound has the structure:

12. the compound of any one of the preceding claims, wherein R⁴Is- (CH)₂)_nOH。

13. The compound of any one of the preceding claims, wherein R⁴Is- (CH)₂)₂OH。

14. The compound of any one of the preceding claims, wherein R⁴Is that

15. The compound of any one of the preceding claims, wherein R⁴Is that

16. The compound of any one of the preceding claims, wherein n2 is 2 or 4.

17. A compound selected from:

18. an empty lipid nanoparticle (empty LNP) comprising a compound of any preceding claim, a phospholipid, a structural lipid and a PEG lipid.

19. The empty LNP of any one of the preceding claims, comprising from about 40 mol% to about 60 mol% of the compound; about 0 mol% to about 20 mol% phospholipid; about 30 mol% to about 50 mol% structural lipid; and about 0 mol% to about 5 mol% PEG lipid.

20. The empty LNP of any preceding claim, wherein the phospholipid is selected from the group consisting of:

1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC),

1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC),

1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

1, 2-di-undecanoyl-sn-glycero-phosphocholine (DUPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),

1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diethyl PC),

1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (ochemsPC),

1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC),

1, 2-di-linolenoyl-sn-glycero-3-phosphocholine,

1, 2-dithienoyl-sn-glycero-3-phosphocholine,

1, 2-docosahexenoyl-sn-glycero-3-phosphocholine, 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE),

1, 2-distearoyl-sn-glycero-3-phosphoethanolamine,

1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine,

1, 2-di-linolenoyl-sn-glycerol-3-phosphoethanolamine,

1, 2-di-arachidonoyl-sn-glycerol-3-phosphoethanolamine,

1, 2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine,

1, 2-dioleoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof.

21. The empty LNP of any one of the preceding claims, wherein the structural lipids are selected from the group consisting of: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, and mixtures thereof.

22. The empty LNP of any one of the preceding claims, wherein the PEG lipid is selected from the group consisting of: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

23. The empty LNP of any of the preceding claims, wherein the PEG lipid is selected from PEG_2k-DMG and PEG-1:

and mixtures thereof.

24. A lipid nanoparticle Loaded (LNP) comprising empty LNP of any preceding claim and one or more therapeutic and/or prophylactic agents.

25. The loaded LNP of any one of the preceding claims, wherein the one or more therapeutic and/or prophylactic agents is a nucleic acid.

26. The loaded LNP of any preceding claim, wherein said nucleic acid is RNA, and wherein said RNA is selected from the group consisting of: short interfering RNA (sirna), asymmetric interfering RNA (airna), RNA interference (RNAi) molecules, microrna (mirna), antagomir, antisense RNA, ribozymes, Dicer-substrate RNA (dsrna), small hairpin RNA (shrna), messenger RNA (mrna), and mixtures thereof.

27. The loaded LNP of any preceding claim, wherein the RNA is mRNA.

28. A pharmaceutical composition comprising the loaded LNP of any one of the preceding claims and a pharmaceutically acceptable carrier.

29. A method of delivering a therapeutic and/or prophylactic agent to a cell in a subject, the method comprising administering to the subject the loaded LNP of any preceding claim.

30. A method of specifically delivering a therapeutic and/or prophylactic agent to an organ of a subject, the method comprising administering the loaded LNP of any preceding claim to the subject.

31. A method of producing a polypeptide of interest in a cell in a subject, the method comprising administering the loaded LNP of any preceding claim to the subject.

32. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the loaded LNP of any preceding claim.